Spectroscopic and analytical characterization of the distribution of iron in intact mitochondria from Saccharomyces cerevisiae

Electron paramagnetic resonance (EPR) and Mössbauer spectroscopy were used to examine the distribution of iron in mitochondria from Saccharomyces cerevisiae. These organelles were packed into EPR and Mössbauer cuvettes, affording spectra with unprecedented signal/noise ratios. EPR spectra of as-isolated intact mitochondria exhibited fourteen distinct signals, some of which were assigned according to previously reported g-values obtained using isolated proteins. Signals from adventitious manganese (II) and iron (III) were largely removed when mitochondria were isolated in buffers supplemented with the metal chelators EDTA or EGTA. Signals were simulated and intensities were quantified to afford spin concentrations and estimates of the concentration of EPR-active species in mitochondria. The effects of treating samples with chemical modifiers were examined. Packed samples were analyzed for protein and metal content, affording averaged values of 50 mg/mL [protein], 590 õM [Fe], 340 õM [Cu], and 17 õM [Mn]. 57Fe-enriched intact mitochondria isolated in the presence of metal chelators exhibited Mössbauer spectra dominated by three components. Approximately 60% of the 57Fe in the sample gave rise to a quadrupole doublet, most of which was diamagnetic. The parameters of this doublet are typical of S = 0 [4Fe-4S]2+ clusters and S = 0 ferrous heme groups. Spectra of samples reduced with dithionite, pH 8.5, suggested that at least half of this doublet arose from [4Fe-4S]2+ clusters. The second major component exhibited in the Mössbauer spectra arose from high-spin ferrous ions (10%-30%). The third major component (15%) came from iron exhibiting magnetic hyperfine interactions and is likely reflected in the Fe-containing species observed by EPR. The results presented here suggest that mitochondria contain ~ 600 õM of Fe overall, ~ 200 â 400 õM organized as [4Fe-4S]2+ clusters, with about 25 õM due to the [4Fe-4S]2+ cluster of aconitase. Approximately 60 õM â 200 õM of the Fe in mitochondria is high-spin ferrous ions, ~ 40 õM as the Rieske S = 1/2 [2Fe-2S]+ cluster of cytochrome bc1, and ~20 õM as the S = 1/2 [2Fe-2S]+ cluster of succinate dehydrogenase. The high-spin ferric hemes of the a3:CuB site of cytochrome oxidase and cytochrome c peroxidase each account for ~ 4 õM of Fe

The concept of quantum sensing from a chemical point of view - Investigations on sensor optimization and application-relevant implementations

XIII, 191 Seite

EPR spectroscopy of antiferromagnetically-coupled Cr3+ molecular wheels

Currently, there is interest in the development of molecular-scale devices for use in quantum information processing (QIP). With this application in mind, physical studies on antiferromagnetically coupled molecular wheels [Cr7MF3(Etglu)(O2CtBu)15(phpy)], where M is a divalent metal cation (M = Mn2+, Zn2+, Ni2+) have been pursued. The heterometallic wheels contain an octagon of metal centres, which are bridged by fluoride ions, pivalate groups and a chiral N-ethyl-D-glutamine molecule which is penta-deprotonated and bound to the metal sites through all available O-donors. They are deep purple in colour and they have been named purple-Cr7M. There is antiferromagnetic coupling between adjacent metal centres, J » -8 cm-1, resulting in a non-zero net spin ground state. The spin-Hamiltonian parameters of this family have been determined.At the heterometal site of purple-Cr7M wheels there is a terminal ligand which can be substituted for a variety of N-donor organic ligands. A series of bidentate N-donor linkers has been used to link Cr7Ni wheels (each wheel Seff = 1/2) to create prototype two-qubit systems. Multi-frequency EPR spectroscopy and SQUID magnetometry has been used to extract the spin-Hamiltonian parameters of this family. It has been shown that the single wheels can be linked together electronically as well as chemically. It has been found that for the unsaturated linkers, there is a weaker interaction between Cr7Ni wheels when longer linkers are used. The strength of interaction is smaller for the saturated linkers than for the unsaturated linkers.The formation of 'green'-Cr7M wheels is different, being templated around a cation. Two new types of wheels have been studied: [tBuCONHC6H12NH2C6H12NHCOtBu][Cr7M2+F8(O2CtBu)16] and [Cs?Cr7MF8(O2CtBu)16]·0.5MeCN (where, M = Mn2+, Zn2+, Ni2+), where the former is templated around a long dialkylammonium group and the latter around a caesium cation. The effect of the templating cation on spectroscopic properties has been determined.Physical studies on a family of antiferromagnetically-coupled homometallic clusters have been pursued. They consist of cyclic arrays of homometallic Cr3+ ions in either a octametallic wheel or hexametallic horseshoes. The horseshoes have the general formula: [CrxFx+5L2x-2]n3- (where L = carboxylate). Cr3+ centres are bridged by pivalate groups and fluorides, while Cr3+ centres at the ends of the chain have terminal fluorides completing their coordination sphere. These terminal fluoride groups are labile enough to be substituted, e.g. [EtNH2][Cr6F7(O2CtBu)10(acac)2] is the product of a substitution reaction with acetylacetone.EThOS - Electronic Theses Online ServiceEPSRCGBUnited Kingdo

Comprehensive Studies of Magnetic Properties of Metal-Organic Frameworks and Molecular Compounds

Single-ion magnets (SIMs) are at the forefront of molecular electronic spin magnets with potential applications in magnetic memory storage devices. However, the magnetic properties of the SIMs are yet to be completely understood, especially the magnetic properties of large anisotropy systems. A part of this dissertation is to utilize optical and neutron spectroscopies such as far-IR magneto-spectroscopy (FIRMS) and inelastic neutron scattering (INS) to quantify the anisotropy and study the phonon properties of the SIMs as two-dimensional (2-D) metal-organic frameworks (MOFs) or coordination polymer (CP), and a molecular magnet. In addition, ab initio calculations are used to understand the origin of the anisotropy and the electronic structure of the systems. Furthermore, the systems studied in this dissertation can also be quantum bit (qubit) candidates. Qubits are the building blocks of quantum computers. The properties of qubits can be determined using pulsed electron paramagnetic resonance (pulsed EPR). The results yielded the spin-lattice relaxation time and the spin-spin relaxation time, where both relaxation times are crucial in determining the effectiveness of the qubit candidates. The second part of this dissertation focuses on studying the symmetry-protected topological states of a Haldane one-dimensional (1-D) spin-1 chain as a 2-D MOF. The topological properties of the Haldane spin-1 chain can be highlighted by the Haldane energy gap that exists between the non-magnetic singlet ground state and the triplet excited state, the fractionalized edge states, and the system’s robustness to external perturbations through symmetry-protection. Optical and neutron spectroscopies in addition to the magnetic susceptibility measurements were used to quantify the energy gaps as well as the anisotropy that governs the system. Furthermore, the spin chain is found to exhibit a critical field and critical temperature where the system observes a phase transition. These studies in this dissertation, in part, aim to give a complete understanding of the magnetic anisotropy and phonon properties of the SIM and qubit systems as well as to have a comprehensive understanding of the topological properties of the Haldane 1-D spin-1 chain system

Spin Labeling with Nitroxide, Trityl, and Copper Labels for Protein Structure Elucidation by EPR Spectroscopy

In this thesis, the intricate challenges that emerge in the application of site-directed spin labeling (SDSL) techniques for structural investigation of biomolecules are addressed, with a specific focus on nitroxide, trityl and copper spin labels. The primary objective revolves around standardizing procedures to enhance the reliability and reproducibility of these SDSL techniques. Furthermore, the application scope of the different spin labels for pulsed dipolar EPR spectroscopy is discussed. In the first section of this thesis, through a comprehensive SDSL strategy utilizing the Yersinia outer protein O (YopO) as a model system, a set of standardized guidelines for SDSL of proteins employing the widely utilized MTSL spin label is developed. Designed as a multi-laboratory benchmark test, the reproducibility and robustness of data acquisition and analysis on the spin-labeled proteins are evaluated and discussed. In the second section of this thesis, a reliable and reproducible spin-labeling protocol for proteins using trityl spin labels is developed. Through meticulous adjustments and fine-tuning of the labeling conditions, the developed protocol sufficiently suppresses aggregation and over labeling of the proteins and enables site-selective spin labeling using maleimide-functionalized trityl spin labels. Subsequently, the trityl-labeled proteins are compared with regards to their EPR sensitivity and the width of the PDS-derived distance distributions. Furthermore, the feasibility of EPR distance measurements at nanomolar concentrations and within cellular systems is assessed. In the first two sections, ambiguous distance distributions were obtained using both nitroxide and trityl spin labels which suggested two distinct conformations of YopO's a-helical backbone. Therefore, in the third section of this thesis, the conformationally restricted bipedal double histidine motif loaded with paramagnetic copper(II) nitrilotriacetic acid (dHis-Cu2+(NTA)) was employed to distinguish between label and protein conformations. Through its reduced conformational flexibility, it was revealed that the a-helical backbone of YopO adopts a single conformation in solution. The herein presented results provide valuable guidelines to the EPR community as well as non-experts for the application of nitroxide spin labels and PDS-EPR in structural biology, outline a reliable protocol for the routine application of maleimide-functionalized trityl spin labels in PDS-EPR, and showcases an approach to differentiate between spin label and protein conformations using the dHis-Cu2+(NTA) spin label