A time-resolved electron paramagnetic resonance spectroscopy study of paramagnetic porphyrins /

There is considerable interest in intramolecular energy transfer, especially in complexes which absorb visible light, because it is crucial to the better understanding of photoharvesting systems in photosynthetic organisms and for utilizing solar energy as well. Porphyrin dimers represent one of the best systems for the exploration of light-induced intramolecular energy transfer. Many kinds of porphyrins and porphyrin dimers have been studied over the past decade, however little attention has been paid to the influence of paramagnetic metals on the behavior of their excited states. In this thesis, Electron Paramagnetic Resonance Spectroscopy (EPR) is used to study such compounds. After light irradiation, porphyrins easily produce a variety of excited states, which are spin polarized and can be detected by the time-resolved (TR) EPR technique. The spin polarized results for vanadyl porphyrins, their electrostatically-coupled dimers, a covalently-linked copper porphyrin-free base porphyrin dimer, and free base porphyrins are presented in this thesis. From these results we can conclude that the spin polarization patterns of vanadyl porphyrins come primarily from the trip-quartet state generated by intersystem crossing (lSC) from the excited sing-doublet state through the trip-doublet state. The spin polarization pattern of electrostatically-coupled vanadyl porphyrin-free base porphyrin dimer is produced by the triplet state of the free base porphyrin half which is coupled to the unpaired electron on the vanadyl ion.

Orientation of Non-Native 2-Monosubstituted and 2,3-Disubstituted 1,4-Naphthoquinones in the A1 binding site of PSI and the effect on the rate of electron transfer : an electron paramagnetic resonance spectroscopy study

The proce-ss ofoxygenic photosynthesis is vital to life on Earth. the central event in photosynthesis is light induced electron transfer that converts light into energy for growth. Ofparticular significance is the membrane bound multisubunit protein known as Photosystem I (PSI). PSI is a reaction centre that is responsible for the transfer of electrons across the membrane to reduce NADP+ to NADPH. The recent publication ofa high resolution X-ray structure of PSI has shown new information about the structure, in particular the electron transfer cofactors, which allows us to study it in more detail. In PSI, the secondary acceptor is crucial for forward electron transfer. In this thesis, the effect of removing the native acceptor phylloquinone and replacing it with a series of structurally related quinones was investigated via transient electron paramagnetic resonance (EPR) experiments. The orientation of non native quinones in the binding site and their ability to function in the electron transfer process was determined. It was found that PSI will readily accept alkyl naphthoquinones and anthraquinone. Q band EPR experiments revealed that the non-native quinones are incorporated into the binding site with the same orientation of the headgroup as in the native system. X band EPR spectra and deuteration experiments indicate that monosubstituted naphthoquinones are bound to the Al site with their side group in the position occupied by the methyl group in native PSI (meta to the hydrogen bonded carbonyl oxygen). X band EPR experiments show that 2, 3- disubstituted methyl naphthoquinones are also incorporated into the Al site in the same orientation as phylloquinone, even with the presence of a halogen- or sulfur-containing side chain in the position normally occupied by the phytyl tail ofphylloquinone. The exception to this is 2-bromo-3-methyl --.- _. -. - -- - - 4 _._ _ _ - _ _ naphthoquinone which has a poorly resolved spectrum, making determination of the orientation difficuh. All of the non-native quinones studied act as efficient electron acceptors. However, forward electron transfer past the quinone could only be demonstrated for anthraquinone, which has a more negative midpoint potential than phylloquinone. In the case of anthraquinone, an increased rate of forward electron transfer compared to native PSI was found. From these results we can conclude that the rate ofelectron transfer from Al to Fx in native PSI lies in the normal region ofthe Marcus Curve.

Transient and Pulsed Electron Paramagnetic Resonance Spectroscopy on Type I Photosynthetic Reaction Centers

Two time-resolved EPR techniques, have been used to study the light induced electron transfer(ET) in Type I photosynthetic reaction centers(RCs). First, pulsed EPR was used to compare PsaA-M688H and PsaB-M668H mutants of Chlamydomonas reinhardtii and Synechosystis sp. PCC 6803.The out-of-phase echo modulation curves combined with other EPR and optical data show that the effect of the mutations is species dependent. Second, transient and pulsed EPR data are presented which show that PsaA-A660N and PsaB-A640N mutations in C. reinhardtii alter the relative quantum yield of ET in the A- and B-branches of PS I. Third, transient EPR studies on RCs from Heliobacillus mobilis that have been exposed to oxygen show partial inhibition of ET. In the RCs in which ET still occurs, the ET kinetics and EPR spectra show evidence of oxidation of some but not all of the, BChl g and BChl g' to Chl a.

Electron Transfer Involving the Phylloquinone (A1) Cofactor of Photosystem I Examined with Time Resolved Absorbance and Electron Paramagnetic Resonance Spectroscopy

The dependence of the electron transfer (ET) rate on the Photosystem I (PSI) cofactor phylloquinone (A1) is studied by time-resolved absorbance and electron paramagnetic resonance (EPR) spectroscopy. Two active branches (A and B) of electron transfer converge to the FX cofactor from the A1A and A1B quinone. The work described in Chapter 5 investigates the single hydrogen bond from the amino acid residue PsaA-L722 backbone nitrogen to A1A for its effect on the electron transfer rate to FX. Room temperature transient EPR measurements show an increase in the rate for the A1A- to FX for the PsaA-L722T mutant and an increased hyperfine coupling to the 2-methyl group of A1A when compared to wild type. The Arrhenius plot of the A1A- to FX ET in the PsaA-L722T mutant suggests that the increased rate is probably the result of a slight change in the electronic coupling between A1A- and FX. The reasons for the non-Arrhenius behavior are discussed. The work discussed in Chapter 6 investigates the directionality of ET at low temperature by blocking ET to the iron-sulfur clusters FX, FA and FB in the menB deletion mutant strain of Synechocyctis sp. PCC 6803, which is unable to synthesize phylloquinone, by incorporating the high midpoint potential (49 mV vs SHE) 2,3-dichloro-1,4-naphthoquinone (Cl2NQ) into the A1A and A1B binding sites. Various EPR spectroscopic techniques were implemented to differentiate between the spectral features created from A and B- branch electron transfer. The implications of this result for the directionality of electron transfer in PS I are discussed. The work discussed in Chapter 7 was done to study the dependence of the heterogeneous ET at low temperature on A1 midpoint potential. The menB PSI mutant contains plastiquinone-9 in the A1 binding site. The solution midpoint potential of the quinone measures 100 mV more positive then wild-type phylloquinone. The irreversible ET to the terminal acceptors FA and FB at low temperature is not controlled by the forward step from A1 to FX as expected due to the thermodynamic differences of the A1 cofactor in the two active branches A and B. Alternatives for the ET heterogeneity are discussed.

Investigation of Frustrated Quasi-One-Dimensional Quantum Spin-Chain Materials

Copper arsenite CuAs2O4 and Copper antimonite CuSb2O4 are S=1/2 (Cu2+ 3d9 electronic configuration) quasi-one-dimensional quantum spin-chain compounds. Both compounds crystallize with tetragonal structures containing edge sharing CuO6 octahedra chains which experience Jahn-Teller distortions. The basal planes of the octahedra link together to form CuO2 ribbon-chains which harbor Cu2+ spin-chains. These compounds are magnetically frustrated with competing nearest-neighbour and next-nearest-neighbour intrachain spin-exchange interactions. Despite the similarities between CuAs2O4 and CuSb2O4, they exhibit very different magnetic properties. In this thesis work, the physical properties of CuAs2O4 and CuSb2O4 are investigated using a variety of experimental techniques which include x-ray diffraction, magnetic susceptibility measurements, heat capacity measurements, Raman spectroscopy, electron paramagnetic resonance, neutron diffraction, and dielectric capacitance measurements. CuAs2O4 exhibits dominant ferromagnetic nearest-neighbour and weaker antiferromagnetic next-nearest-neighbour intrachain spin-exchange interactions. The ratio of the intrachain interactions amounts to Jnn/Jnnn = -4.1. CuAs2O4 was found to order with a ferromagnetic groundstate below TC = 7.4 K. An extensive physical characterization of the magnetic and structural properties of CuAs2O4 was carried out. Under the effect of hydrostatic pressure, CuAs2O4 was found to undergo a structural phase transition at 9 GPa to a new spin-chain structure. The structural phase transition is accompanied by a severe alteration of the magnetic properties. The high-pressure phase exhibits dominant ferromagnetic next-nearest-neighbour spin-exchange interactions and weaker ferromagnetic nearest-neighbour interactions. The ratio of the intrachain interactions in the high-pressure phase was found to be Jnn/Jnnn = 0.3. Structural and magnetic characterizations under hydrostatic pressure are reported and a relationship between the structural and magnetic properties was established. CuSb2O4 orders antiferromagnetically below TN = 1.8 K with an incommensurate helicoidal magnetic structure. CuSb2O4 is characterized by ferromagnetic nearest-neighbour and antiferromagnetic next-nearest-neighbour spin-exchange interactions with Jnn/Jnnn = -1.8. A (H, T) magnetic phase diagram was constructed using low-temperature magnetization and heat capacity measurements. The resulting phase diagram contains multiple phases as a consequence of the strong intrachain magnetic frustration. Indications of ferroelectricity were observed in the incommensurate antiferromagnetic phase.

The finite bandwidth model for spin fluctuations in Pd

The frequency dependence of the electron-spin fluctuation spectrum, P(Q), is calculated in the finite bandwidth model. We find that for Pd, which has a nearly full d-band, the magnitude, the range, and the peak frequency of P(Q) are greatly reduced from those in the standard spin fluctuation theory. The electron self-energy due to spin fluctuations is calculated within the finite bandwidth model. Vertex corrections are examined, and we find that Migdal's theorem is valid for spin fluctuations in the nearly full band. The conductance of a normal metal-insulator-normal metal tunnel junction is examined when spin fluctuations are present in one electrode. We find that for the nearly full band, the momentum independent self-energy due to spin fluctuations enters the expression for the tunneling conductance with approximately the same weight as the self-energy due to phonons. The effect of spin fluctuations on the tunneling conductance is slight within the finite bandwidth model for Pd. The effect of spin fluctuations on the tunneling conductance of a metal with a less full d-band than Pd may be more pronounced. However, in this case the tunneling conductance is not simply proportional to the self-energy.

Developing a numerical inverse-theory-based extraction of orientation-dependent relaxation rates from partially- relaxed spectra

Second-rank tensor interactions, such as quadrupolar interactions between the spin- 1 deuterium nuclei and the electric field gradients created by chemical bonds, are affected by rapid random molecular motions that modulate the orientation of the molecule with respect to the external magnetic field. In biological and model membrane systems, where a distribution of dynamically averaged anisotropies (quadrupolar splittings, chemical shift anisotropies, etc.) is present and where, in addition, various parts of the sample may undergo a partial magnetic alignment, the numerical analysis of the resulting Nuclear Magnetic Resonance (NMR) spectra is a mathematically ill-posed problem. However, numerical methods (de-Pakeing, Tikhonov regularization) exist that allow for a simultaneous determination of both the anisotropy and orientational distributions. An additional complication arises when relaxation is taken into account. This work presents a method of obtaining the orientation dependence of the relaxation rates that can be used for the analysis of the molecular motions on a broad range of time scales. An arbitrary set of exponential decay rates is described by a three-term truncated Legendre polynomial expansion in the orientation dependence, as appropriate for a second-rank tensor interaction, and a linear approximation to the individual decay rates is made. Thus a severe numerical instability caused by the presence of noise in the experimental data is avoided. At the same time, enough flexibility in the inversion algorithm is retained to achieve a meaningful mapping from raw experimental data to a set of intermediate, model-free

Extracting Ramachandran torsional angle distributions from 2D NMR data using Tikhonov regularization /

Solid state nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for studying structural and dynamical properties of disordered and partially ordered materials, such as glasses, polymers, liquid crystals, and biological materials. In particular, twodimensional( 2D) NMR methods such as ^^C-^^C correlation spectroscopy under the magicangle- spinning (MAS) conditions have been used to measure structural constraints on the secondary structure of proteins and polypeptides. Amyloid fibrils implicated in a broad class of diseases such as Alzheimer's are known to contain a particular repeating structural motif, called a /5-sheet. However, the details of such structures are poorly understood, primarily because the structural constraints extracted from the 2D NMR data in the form of the so-called Ramachandran (backbone torsion) angle distributions, g{^,'4)), are strongly model-dependent. Inverse theory methods are used to extract Ramachandran angle distributions from a set of 2D MAS and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) data. This is a vastly underdetermined problem, and the stability of the inverse mapping is problematic. Tikhonov regularization is a well-known method of improving the stability of the inverse; in this work it is extended to use a new regularization functional based on the Laplacian rather than on the norm of the function itself. In this way, one makes use of the inherently two-dimensional nature of the underlying Ramachandran maps. In addition, a modification of the existing numerical procedure is performed, as appropriate for an underdetermined inverse problem. Stability of the algorithm with respect to the signal-to-noise (S/N) ratio is examined using a simulated data set. The results show excellent convergence to the true angle distribution function g{(j),ii) for the S/N ratio above 100.

Chelated fluoroboron cations : synthesis and multinuclear nmr studies

The preparation of chelated difluoroboron cations (DD)BF2+, where DD is a saturated polydentate tertiary-amine or polydentate aromatic ligand, has been systematically studied by using multinuclear solution and solid state nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Three new methods of synthesis of (DD)BF2+ cations are reported, and compared with the previous method of reacting a chelating donor with Et20.BF3. The methods most effective for aromatic donors such as 1,1O-phenanthroline are ineffective for saturated polydentate tertiary-amines like N,N,N' ,Nil ,Nil-pentamethyldiethylenetriamine. Polydentate tertiary-amine donors that form 5-membered rings upon bidentate chelation were found to chelate effectively when the BF2 source contained two leaving groups (a heavy halide and a Lewis base such as pyridine =pyr or isoxazole =ISOX), i.e., pyr.BF2X (X = CI or Br), ISOX.BF2X and (pyr)2BF2+. Those that would form 6membered rings upon chelation do not chelate by any of the four methods. Polydentate aromatic ligands chelate effectively when the BF2 source contained a weak Lewis base, e.g., ISOX.BF3, ISOX.BF2X and Et20.BF3. Bidentate chelation by polydentate tertiaryamine and aromatic donors leads to nmr parameters that are significantly different then their (D)2BF2+ relatives (D =monod~ntate t-amines or pyridines). The chelated haloboron cations (DD)BFCI+, and (DD)BFBr+ were generated from D.BFX2 adducts for all ligands that form BF2+ cations above. In addition, the (DD)BCI2+ and (DD)BBr2+ cations were formed from D.BX3 adducts by the chelating aromatic ligands, except for the aromatic ligand 1,8-bis(dimethylamino)naphthalene, which formed only the (DD)BF2+ cation, apparently due to its extreme steric hindrance. Chelation by a donor is a two-step reaction. For polydentate tertiary-amine ligands, the two rates appear to be very dependent on the two possible leaving groups on the central boron atom. The order of increasing ease of displacement for the donors was: pyr < Cl < Br < ISOX. The rate of chelation by polydentate aromatic ligands appears to be dependent on the displacement of the first ligand from the boron. The order of increasing ease of displacement for the donors was: pyr < CI < ISOX ~ Br < Et20.

NMR studies of mixed tetrahaloborates and some related boron trihalide complexes /|nGary John Schrobilgen. -- 260 St. Catharines, Ont. : [s. n.],

Nuclear magnetic resonance spectroscopy has been used to study donor-acceptor complexes of boron trifluoride with several ureas, tetramethylthiourea, tetramethylselenourea, and tetramethylquanidine as well as adducts of tetramethyl- -urea with BF2Cl, BFC1 2 , and BC1 3 - A large number of mixed tetrahaloborate ions, including some of the ternary ones such as BF2CIBr-,have been obtained by ligand exchange reactions and studied by NMR techniques. The bonding in these ions is of the same inherent interest as the bonding in the isoelectronic tetrahalomethanes which have been the subject of many detailed studies and have been involved in a controversy concerning the existence of and the nature of "fluorine hyperconjugation" or C-F P1T- Pn bonding_ Ligand exchange reactions also gave rise to the difluoroboron cation, (TMU)20BF2+o The difluoroboron cation has been observed in solutions of TMU-BF3 , and has been proposed as a possible intermediate for fluorine exchange reactions in BF3 adducts.

NMR studies of strong hydrogen bond to fluoride ion

Systems such as MF/diol (M = alkali metal) and }1F/carboxylic acid were subjected to IH, I9F and 13C nmr study to investigate the nature of the very strong H-bonding of fluoride ions with these systems. Evidence indicates a strong H-bond in diol-fluoride systems (~H ~ -(56) kJ mol-I) which is stronger than most 'typical' H-bonds (~H = -(12-40) kJ mol-I), but weaker than that reported for carboxylic acid-fluoride systems (~H ~ -(120) kJ mol-I). Approximate fluoride H-bonded shifts (o(OH)OHF) were evaluated for MF/diol systems from IH chemical shift measurements. No direct correlation was observed between I9F chemical shift and H-bond strength. Thermodynamic parameters were calculated from temperature dependent IH and 19F shifts. Preliminary studies of BUn 4NF-acetylacetone by I9F nmr were conducted at low temperatures and a possible Jmax (ca. 400 Hz) is reported for the fluoride ion H-bonded to acetylacetone. Highfield shift for non-protonated carbons and downfield shift for protonated carbons were observed in carboxylic acid/KF systems. Significant decreas$in I3C TI due to strong H-bonding to fluoride ions were also detected in both diol and carboxylic acid systems. Anomalous results were obtained, such as increasing NOE with increasing temperature in neat 1,2-ethanediol (values above the theoretical maximum of 1.988) and in 1,2-ethanediol/KF. The large 13C NOE's for carboxy carbons in neat carboxylic acids which are. further enhanced by the addition of KF are also unusual.

Simultaneous extraction of order parameters and orientational distribution fuctions from 31[superscript]P NMR spectra of magnetically partially oriented phospholipid bilayers

Order parameter profiles extracted from the NMR spectra of model membranes are a valuable source of information about their structure and molecular motions. To al1alyze powder spectra the de-Pake-ing (numerical deconvolution) ~echnique can be used, but it assumes a random (spherical) dist.ribution of orientations in the sample. Multilamellar vesicles are known to deform and orient in the strong magnetic fields of NMR magnets, producing non-spherical orientation distributions. A recently developed technique for simultaneously extracting the anisotropies of the system as well as the orientation distributions is applied to the analysis of partially magnetically oriented 31p NMR spectra of phospholipids. A mixture of synthetic lipids, POPE and POPG, is analyzed to measure distortion of multilamellar vesicles in a magnetic field. In the analysis three models describing the shape of the distorted vesicles are examined. Ellipsoids of rotation with a semiaxis ratio of about 1.14 are found to provide a good approximation of the shape of the distorted vesicles. This is in reasonable agreement with published experimental work. All three models yield clearly non-spherical orientational distributions, as well as a precise measure of the anisotropy of the chemical shift. Noise in the experimental data prevented the analysis from concluding which of the three models is the best approximation. A discretization scheme for finding stability in the algorithm is outlined

NMR characterization of chlorhexidine in lipid-based formulations /

A mixture of Chlorhexidine digluconate (CHG) with glycerophospholipid 1,2-dimyristoyl- <^54-glycero-3-phospocholine (DMPC-rf54) was analysed using ^H nuclear magnetic resonance. To analyze powder spectra, the de-Pake-ing technique was used. The method is able to extract simultaneously both the orientation distribution function and the anisotropy distribution function. The spectral moments, average order parameter profiles, and longitudinal and transverse relaxation times were used to explore the structural phase behaviour of various DMPC/CHG mixtures in the temperature range 5-60°C.

Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems.

Cyclotetrasiloxane Frameworks for the Chemoenzymatic Synthesis of Oligoesters

Immobilized lipase B from Candida antarctica (Novozym® 435, N435) was utilized as part of a chemoenzymatic strategy for the synthesis of branched polyesters based on a cyclotetrasiloxane core in the absence of solvent. Nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry were utilized to monitor the reactions between tetraester cyclotetrasiloxanes and aliphatic diols. The enzyme-mediated esterification reactions can achieve 65– 80% consumption of starting materials in 24–48 h. Longer reaction times, 72–96 h, resulted in the formation of cross-linked gel-like networks. Gel permeation chromatography of the polymers indicated that the masses were Mw ¼ 11 400, 13 100, and 19 400 g mol 1 for the substrate pairs of C7D4 ester/ octane-1,8-diol, C10D4 ester/pentane-1,5-diol and C10D4 ester/octane-1,8-diol respectively, after 48 h. Extending the polymerization for an additional 24 h with the C10D4 ester/octane-1,8-diol pair gave Mw ¼ 86 800 g mol 1. To the best of our knowledge this represents the first report using lipase catalysis to produce branched polymers that are built from a cyclotetrasiloxane core.