Forbidden transitions are not observed in the continuous-wave electron paramagnetic resonance (EP... more Forbidden transitions are not observed in the continuous-wave electron paramagnetic resonance (EPR) spectrum nor in the free induction decay because, unlike allowed transitions, their coherences have no observable magnetic moment and are spectroscopically silent. Yet, the paramagnetic relaxation described by Redfield theory can cause coherence transfer between any types of transitions. Coherence transfer between allowed transitions is now known to cause noticeable changes in EPR spectra, but coherence transfer involving forbidden transitions has long been considered to be negligible because those coherences are silent and unseen. However, our simulations of a simple model system indicate that coherence transfer with silent transitions can introduce new features into EPR spectra. The EPR-silent coherence of a forbidden transition can be transferred to an allowed transition by paramagnetic relaxation. A silent coherence can have consequences felt in the EPR spectrum. Dedicated to Professor James Hyde on the occasion of his 85th birthday. He has demonstrated many times that it is possible to measure very subtle effects provided they can be encoded or modulated to distinguish them from other responses and noise.
Hyperfine sublevel correlation (HYSCORE) spectra of paramagnetic centers that have nuclei with nu... more Hyperfine sublevel correlation (HYSCORE) spectra of paramagnetic centers that have nuclei with nuclear spin I) 1 and isotropic hyperfine interactions (HFIs) and arbitrary nuclear quadrupole interactions (NQIs) are shown to consist of ridges that have zero width. A parametric presentation of these ridges is suggested that shows the range of possible frequencies in the HYSCORE spectrum and aids in spectral assignments and rapid estimation of spin Hamiltonian parameters. An alternative approach for the spectral density calculation is presented. This methodology is based on spectral decomposition of the Hamiltonian and requires only the eigenvalues. An atlas of HYSCORE spectra is given in the Supporting Information for this paper. This approach is applied to the estimation of the spin Hamiltonian parameters of the oxovanadium-EDTA complex.
The properties and dynamics of isotropic electron spins are drastically simpler than for anisotro... more The properties and dynamics of isotropic electron spins are drastically simpler than for anisotropic spin systems with S = 1/2. The classical rotating coordinate frame with axes moving at the microwave frequency provides convenient descriptions of isotropic electron spins, their magnetic moments and their magnetic resonance behavior but does not provide a suitable description for paramagnetic centers having significant anisotropy in their $$\overset{\lower0.5em\hbox{$\smash{\scriptscriptstyle\leftrightarrow}$}} {g}$$ g ↔ matrix. The relative merits of several different frames are discussed including the laboratory frame, a tilted rotating frame and effective field frames. It is seen that simulations of the anisotropic spins are more easily performed with a different frame for each orientation of the spin, but results can be readily visualized in the laboratory frame.
Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study... more 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.
The unexpected formation of a highly strained polycyclic amine was found in a one-pot synthesis f... more The unexpected formation of a highly strained polycyclic amine was found in a one-pot synthesis from cyclopentanone, dimethylfumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically-shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide was studied in solution. The spin relaxation seems well suited for use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to N-O• group.
A conjugated polymer semiconductor was synthesized, which exhibits a high-spin ground state and r... more A conjugated polymer semiconductor was synthesized, which exhibits a high-spin ground state and robust stability.
The journal of physical chemistry. B, Jan 13, 2018
Triarylmethyl (TAM) radicals have become widely used free radicals in the past few years. Their e... more Triarylmethyl (TAM) radicals have become widely used free radicals in the past few years. Their electron spins have long relaxation times and narrow electron paramagnetic resonance (EPR) lines, which make them an important class of probes and tags in biological applications and materials science. In this work, we propose a new approach to characterize librations by means of TAM radicals. The temperature dependence of motional parameter ⟨α⟩τ, where ⟨α⟩ is the mean-squared amplitude of librations and τ is their characteristic time, is obtained by comparison of the 1/ T phase-relaxation rates at X- and Q-band EPR frequencies. We study three soft matrixes, viz., glassy trehalose and two ionic liquids, using TAMs with optimized relaxation properties OX063D and a dodeca- n-butyl homologue of Finland trityl (DBT). The motional parameters ⟨α⟩τ obtained using TAMs are in excellent agreement with those obtained by means of nitroxide radicals. At the same time, the new TAM-based approach has (...
Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They p... more Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-HO-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-br...
In general, a variable repetition rate of the x-ray bunches is needed to explore time domain prob... more In general, a variable repetition rate of the x-ray bunches is needed to explore time domain problems using x-rays. In some instances, the results of several hundreds or thousands of x-ray pulses must be averaged requiring the sample to be in the same time-dependent state each time the monitoring pulse strikes. The important point is that before a second x-ray pulse hits the sample, the system must return to its initial relaxed ground state prior to another spectrum pulse in order to prepare the same excited state again. Our key design principle employs a subsonic, rotating mirror whose period is slaved to the synchrotron intrapulse period. The synchrotron x-ray bunches will be reflected a distance of about 2 m to a narrow 0.5-mm slit just in front of the sample. A mirror rotating at a low 7500 rpm (125 Hz) is sufficient to select a single synchrotron pulse for the Advanced Photon Source. The very precise phase stability required by this method is currently available in rotating mir...
Facile and high-yielding procedures for synthesis of monocarboxylic acid derivatives of triarylme... more Facile and high-yielding procedures for synthesis of monocarboxylic acid derivatives of triarylmethyl radicals (TAMs) were developed. Reaction of methyl thioglycolate with tris(2,3,5,6tetrathiaaryl)methyl cation smoothly afforded the monosubstituted TAM derivative, which was hydrolyzed to a monocarboxylic acid, with the TAM moiety attached to thioglycolic acid via the sulfur atom. Alternatively, the diamagnetic tricarboxylic acid precursor of Finland trityl was transformed to a trimethyl ester and partially hydrolyzed under controlled conditions. The diester product was isolated and the remaining fractions were converted back to the trimethyl ester for production of more diester. The first representatives of TAM biradicals with different TAM cores and interspin distances were obtained by reaction of these new TAM monocaboxylic acids with N,N′-dimethylethylenediamine.
ABSTRACTThe structures of C5H5NiNO in a reversible photochemical reaction were studied via EXAFS,... more ABSTRACTThe structures of C5H5NiNO in a reversible photochemical reaction were studied via EXAFS, FTIR, and optical absorption spectroscopies. A photoexcited intermediate with distinctively different EXAFS, IR, and optical absorption spectra from those of the ground state molecules was generated upon irradiation using 365 nm light at 20K in a 3-methylpentane solution. The reverse reaction was induced by irradiation with 310 nm light. The EXAFS data analysis has shown a 0. 12 Å elongation of the Ni-N bond and the bending of Ni-N-O in the photoexcited intermediate. Several ZINDO calculations were conducted based on the structures obtained from the EXAFS spectroscopy. These calculations reproduced the changes in the optical spectra and the intramolecular electron transfer in C5H5NiNO.
The carotenoid astaxanthin forms novel metal ion complexes with Ca 2+ , Zn 2+ and Fe 2+. MS and N... more The carotenoid astaxanthin forms novel metal ion complexes with Ca 2+ , Zn 2+ and Fe 2+. MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca 2+ and Zn 2+ at low salt concentrations < 0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca 2+ and by a factor of 7 for Zn 2+ as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (> 0.2 mM) of salt 2:1 metal:astaxanthin complexes were formed in ethanol. In the presence of Ca 2+ and Zn 2+ salts the lifetime of the radical cation and dication formed electrochemically decreased relative to that of the carotenoid neutral radical. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical while proton loss at the C5, C9 or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41 or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13-methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.
The direct dimension or time following directly after a pulse sequence in pulse EPR is rarely uti... more The direct dimension or time following directly after a pulse sequence in pulse EPR is rarely utilized. Many of the reasons for this stem from limitations in the technology available at the time pulse EPR was developed. The underutilization of the direct dimension can limit sensitivity, information content, and accurate analysis of data. Some features of the direct dimension are illustrated using two-pulse ESEEM spectroscopy.
DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United St... more DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. '"'"'"""""'"" MASTER by • contractorof the U.S. Go_._mt
The P1 duplex then docks into the pre-folded ribozyme core by forming tertiary contacts. P1 docki... more The P1 duplex then docks into the pre-folded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a pre-folded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1 to 50 ns regime. Using substrates that favor the docked or the undocked states, it was established that the nitroxide was capable of reporting P1 duplex motions. Using these nitroxide labeled substrates, it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state (Grant et.al., 2009, JACS, 131, 3136-7). This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. Work is underway to investigate P1 motions in various mutant ribozymes in order to establish detailed correlations between nanosecond dynamics of P1 with ribozyme tertiary folding and catalytic activity. This may help in understanding the relationship between RNA structure, dynamics, and function.
Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although e... more Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450 eryF. UV/vis absorbance spectra of P450 eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K S) 4 and 200 µM, respectively. The broadening and shifting observed in the 2D-{ 1 H, 15 N}-HSQC-monitored titrations of 15 N-Phe-labeled P450 eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hillequation-derived K S values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15 N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450 eryF. This finding is in agreement with X-ray crystal structures of bound P450 eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the lowaffinity TST molecule or multiple TST-bound P450 eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.
Forbidden transitions are not observed in the continuous-wave electron paramagnetic resonance (EP... more Forbidden transitions are not observed in the continuous-wave electron paramagnetic resonance (EPR) spectrum nor in the free induction decay because, unlike allowed transitions, their coherences have no observable magnetic moment and are spectroscopically silent. Yet, the paramagnetic relaxation described by Redfield theory can cause coherence transfer between any types of transitions. Coherence transfer between allowed transitions is now known to cause noticeable changes in EPR spectra, but coherence transfer involving forbidden transitions has long been considered to be negligible because those coherences are silent and unseen. However, our simulations of a simple model system indicate that coherence transfer with silent transitions can introduce new features into EPR spectra. The EPR-silent coherence of a forbidden transition can be transferred to an allowed transition by paramagnetic relaxation. A silent coherence can have consequences felt in the EPR spectrum. Dedicated to Professor James Hyde on the occasion of his 85th birthday. He has demonstrated many times that it is possible to measure very subtle effects provided they can be encoded or modulated to distinguish them from other responses and noise.
Hyperfine sublevel correlation (HYSCORE) spectra of paramagnetic centers that have nuclei with nu... more Hyperfine sublevel correlation (HYSCORE) spectra of paramagnetic centers that have nuclei with nuclear spin I) 1 and isotropic hyperfine interactions (HFIs) and arbitrary nuclear quadrupole interactions (NQIs) are shown to consist of ridges that have zero width. A parametric presentation of these ridges is suggested that shows the range of possible frequencies in the HYSCORE spectrum and aids in spectral assignments and rapid estimation of spin Hamiltonian parameters. An alternative approach for the spectral density calculation is presented. This methodology is based on spectral decomposition of the Hamiltonian and requires only the eigenvalues. An atlas of HYSCORE spectra is given in the Supporting Information for this paper. This approach is applied to the estimation of the spin Hamiltonian parameters of the oxovanadium-EDTA complex.
The properties and dynamics of isotropic electron spins are drastically simpler than for anisotro... more The properties and dynamics of isotropic electron spins are drastically simpler than for anisotropic spin systems with S = 1/2. The classical rotating coordinate frame with axes moving at the microwave frequency provides convenient descriptions of isotropic electron spins, their magnetic moments and their magnetic resonance behavior but does not provide a suitable description for paramagnetic centers having significant anisotropy in their $$\overset{\lower0.5em\hbox{$\smash{\scriptscriptstyle\leftrightarrow}$}} {g}$$ g ↔ matrix. The relative merits of several different frames are discussed including the laboratory frame, a tilted rotating frame and effective field frames. It is seen that simulations of the anisotropic spins are more easily performed with a different frame for each orientation of the spin, but results can be readily visualized in the laboratory frame.
Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy is a powerful technique for the study... more 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.
The unexpected formation of a highly strained polycyclic amine was found in a one-pot synthesis f... more The unexpected formation of a highly strained polycyclic amine was found in a one-pot synthesis from cyclopentanone, dimethylfumarate and ammonium acetate. This multistep reaction includes 1,3-dipolar cycloaddition of dimethyl fumarate to the cyclic azomethine ylide formed in situ from cyclopentanone and ammonia. The polycyclic amine product was easily converted into a sterically-shielded polycyclic nitroxide. The EPR spectra and spin relaxation behavior of the nitroxide was studied in solution. The spin relaxation seems well suited for use as a biological spin label and are comparable with those of cyclic nitroxides with two spirocyclic moieties adjacent to N-O• group.
A conjugated polymer semiconductor was synthesized, which exhibits a high-spin ground state and r... more A conjugated polymer semiconductor was synthesized, which exhibits a high-spin ground state and robust stability.
The journal of physical chemistry. B, Jan 13, 2018
Triarylmethyl (TAM) radicals have become widely used free radicals in the past few years. Their e... more Triarylmethyl (TAM) radicals have become widely used free radicals in the past few years. Their electron spins have long relaxation times and narrow electron paramagnetic resonance (EPR) lines, which make them an important class of probes and tags in biological applications and materials science. In this work, we propose a new approach to characterize librations by means of TAM radicals. The temperature dependence of motional parameter ⟨α⟩τ, where ⟨α⟩ is the mean-squared amplitude of librations and τ is their characteristic time, is obtained by comparison of the 1/ T phase-relaxation rates at X- and Q-band EPR frequencies. We study three soft matrixes, viz., glassy trehalose and two ionic liquids, using TAMs with optimized relaxation properties OX063D and a dodeca- n-butyl homologue of Finland trityl (DBT). The motional parameters ⟨α⟩τ obtained using TAMs are in excellent agreement with those obtained by means of nitroxide radicals. At the same time, the new TAM-based approach has (...
Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They p... more Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-HO-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-br...
In general, a variable repetition rate of the x-ray bunches is needed to explore time domain prob... more In general, a variable repetition rate of the x-ray bunches is needed to explore time domain problems using x-rays. In some instances, the results of several hundreds or thousands of x-ray pulses must be averaged requiring the sample to be in the same time-dependent state each time the monitoring pulse strikes. The important point is that before a second x-ray pulse hits the sample, the system must return to its initial relaxed ground state prior to another spectrum pulse in order to prepare the same excited state again. Our key design principle employs a subsonic, rotating mirror whose period is slaved to the synchrotron intrapulse period. The synchrotron x-ray bunches will be reflected a distance of about 2 m to a narrow 0.5-mm slit just in front of the sample. A mirror rotating at a low 7500 rpm (125 Hz) is sufficient to select a single synchrotron pulse for the Advanced Photon Source. The very precise phase stability required by this method is currently available in rotating mir...
Facile and high-yielding procedures for synthesis of monocarboxylic acid derivatives of triarylme... more Facile and high-yielding procedures for synthesis of monocarboxylic acid derivatives of triarylmethyl radicals (TAMs) were developed. Reaction of methyl thioglycolate with tris(2,3,5,6tetrathiaaryl)methyl cation smoothly afforded the monosubstituted TAM derivative, which was hydrolyzed to a monocarboxylic acid, with the TAM moiety attached to thioglycolic acid via the sulfur atom. Alternatively, the diamagnetic tricarboxylic acid precursor of Finland trityl was transformed to a trimethyl ester and partially hydrolyzed under controlled conditions. The diester product was isolated and the remaining fractions were converted back to the trimethyl ester for production of more diester. The first representatives of TAM biradicals with different TAM cores and interspin distances were obtained by reaction of these new TAM monocaboxylic acids with N,N′-dimethylethylenediamine.
ABSTRACTThe structures of C5H5NiNO in a reversible photochemical reaction were studied via EXAFS,... more ABSTRACTThe structures of C5H5NiNO in a reversible photochemical reaction were studied via EXAFS, FTIR, and optical absorption spectroscopies. A photoexcited intermediate with distinctively different EXAFS, IR, and optical absorption spectra from those of the ground state molecules was generated upon irradiation using 365 nm light at 20K in a 3-methylpentane solution. The reverse reaction was induced by irradiation with 310 nm light. The EXAFS data analysis has shown a 0. 12 Å elongation of the Ni-N bond and the bending of Ni-N-O in the photoexcited intermediate. Several ZINDO calculations were conducted based on the structures obtained from the EXAFS spectroscopy. These calculations reproduced the changes in the optical spectra and the intramolecular electron transfer in C5H5NiNO.
The carotenoid astaxanthin forms novel metal ion complexes with Ca 2+ , Zn 2+ and Fe 2+. MS and N... more The carotenoid astaxanthin forms novel metal ion complexes with Ca 2+ , Zn 2+ and Fe 2+. MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca 2+ and Zn 2+ at low salt concentrations < 0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca 2+ and by a factor of 7 for Zn 2+ as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (> 0.2 mM) of salt 2:1 metal:astaxanthin complexes were formed in ethanol. In the presence of Ca 2+ and Zn 2+ salts the lifetime of the radical cation and dication formed electrochemically decreased relative to that of the carotenoid neutral radical. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical while proton loss at the C5, C9 or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41 or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13-methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.
The direct dimension or time following directly after a pulse sequence in pulse EPR is rarely uti... more The direct dimension or time following directly after a pulse sequence in pulse EPR is rarely utilized. Many of the reasons for this stem from limitations in the technology available at the time pulse EPR was developed. The underutilization of the direct dimension can limit sensitivity, information content, and accurate analysis of data. Some features of the direct dimension are illustrated using two-pulse ESEEM spectroscopy.
DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United St... more DISCLAIMER This report was prepared as an account of work sponsored by an agency of the United States Government. Neither the United States Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof. '"'"'"""""'"" MASTER by • contractorof the U.S. Go_._mt
The P1 duplex then docks into the pre-folded ribozyme core by forming tertiary contacts. P1 docki... more The P1 duplex then docks into the pre-folded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a pre-folded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1 to 50 ns regime. Using substrates that favor the docked or the undocked states, it was established that the nitroxide was capable of reporting P1 duplex motions. Using these nitroxide labeled substrates, it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state (Grant et.al., 2009, JACS, 131, 3136-7). This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. Work is underway to investigate P1 motions in various mutant ribozymes in order to establish detailed correlations between nanosecond dynamics of P1 with ribozyme tertiary folding and catalytic activity. This may help in understanding the relationship between RNA structure, dynamics, and function.
Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although e... more Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450 eryF. UV/vis absorbance spectra of P450 eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of K S) 4 and 200 µM, respectively. The broadening and shifting observed in the 2D-{ 1 H, 15 N}-HSQC-monitored titrations of 15 N-Phe-labeled P450 eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hillequation-derived K S values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15 N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands "contact" the same phenylalanines within the active site of P450 eryF. This finding is in agreement with X-ray crystal structures of bound P450 eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the lowaffinity TST molecule or multiple TST-bound P450 eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.
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