Search

The performance of a room temperature, zero-field MASER operating at 1.45 GHz has been examined. Nanosecond laser pulses, which are essentially instantaneous on the timescale of the spin dynamics, allow the visible-to-microwave conversion efficiency and temporal response of the MASER to be measured as a function of excitation energy. It is observed that the timing and amplitude of the MASER output pulse are correlated with the laser excitation energy: at higher laser energy, the microwave pulses have larger amplitude and appear after shorter delay than those recorded at lower laser energy. Seeding experiments demonstrate that the output variation may be stabilized by an external source and establish the minimum seeding power required. The dynamics of the MASER emission may be modeled by a pair of first order, non-linear differential equations, derived from the Lotka-Volterra model (Predator-Prey), where by the microwave mode of the resonator is the predator and the spin polarization in the triplet state of pentacene is the prey. Simulations allowed the Einstein coefficient of stimulated emission, the spin-lattice relaxation and the number of triplets contributing to the MASER emission to be estimated. These are essential parameters for the rational improvement of a MASER based on a spin-polarized triplet molecule., Competing Interests: The authors declare no competing financial interests.

Surfaces with built-in antimicrobial activity have the potential to reduce hospital-acquired infections. One promising strategy is to create functionalised surfaces which, following illumination with visible light, are able to generate singlet oxygen under aerobic conditions. In contrast to antibiotics, the mechanism of bacterial kill by species derived from reactions with singlet oxygen is completely unselective, therefore offering little room for evolutionary adaptation. Here we consider five commercially available organic photosensitiser dyes encapsulated in silicone polymer that show varied antimicrobial activity. We correlate density functional theory calculations with UV-Vis spectroscopy, electron paramagnetic resonance spectroscopy and singlet oxygen production measurements in order to define and test the elements required for efficacious antimicrobial activity. Our approach forms the basis for the rational in silico design and spectroscopic screening of simple and efficient self-sterilising surfaces made from cheap, low toxicity photosensitiser dyes encapsulated in silicone.

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation., (© 2016 The Author(s).)

Connexin (Cx) proteins and gap junctions support the formation of neuronal and glial syncytia that are linked to different forms of rhythmic firing and oscillatory activity in the CNS. In this study, quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to profile developmental expression of two specific Cx proteins, namely glial Cx43 and neuronal Cx36, in postnatal lumbar spinal cord aged 4, 7, and 14 days. Extracellular electrophysiology was used to determine the contribution of Cx36 and Cx43 to a previously described form of 4-aminopyridine (4-AP)-induced 4-12 Hz rhythmic activity within substantia gelatinosa (SG) of rat neonatal dorsal horn (DH) in vitro. The involvement of Cx36 and Cx43 was probed pharmacologically using quinine, a specific uncoupler of Cx36 and the mimetic peptide blocker Gap 26 which targets Cx43. After establishment of 4-12 Hz rhythmic activity by 4-AP (25 μmol/L), coapplication of quinine (250 μmol/L) reduced 4-AP-induced 4-12 Hz rhythmic activity (P < 0.05). Preincubation of spinal cord slices with Gap 26 (100 μmol/L), compromised the level of 4-AP-induced 4-12 Hz rhythmic activity in comparison with control slices preincubated in ACSF alone (P < 0.05). Conversely, the nonselective gap junction "opener" trimethylamine (TMA) enhanced 4-12 Hz rhythmic behavior (P < 0.05), further supporting a role for Cx proteins and gap junctions. These data have defined a physiological role for Cx36 and Cx43 in rhythmic firing in SG, a key nociceptive processing area of DH. The significance of these data in the context of pain and Cx proteins as a future analgesic drug target requires further study., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The adaptor protein Grb2 is a key element of mitogenetically important signaling pathways. With its SH2 domain it binds to upstream targets while its SH3 domains bind to downstream proteins thereby relaying signals from the cell membranes to the nucleus. The Grb2 SH2 domain binds to its targets by recognizing a phosphotyrosine (pY) in a pYxNx peptide motif, requiring an Asn at the +2 position C-terminal to the pY with the residue either side of this Asn being hydrophobic. Structural analysis of the Grb2 SH2 domain in complex with its cognate peptide has shown that the peptide adopts a unique β-turn conformation, unlike the extended conformation that phosphopeptides adopt when bound to other SH2 domains. TrpEF1 (W121) is believed to force the peptide into this unusual conformation conferring this unique specificity to the Grb2 SH2 domain. Using X-ray crystallography, electron paramagnetic resonance (EPR) spectroscopy, and isothermal titration calorimetry (ITC), we describe here a series of experiments that explore the role of TrpEF1 in determining the specificity of the Grb2 SH2 domain. Our results demonstrate that the ligand does not adopt a pre-organized structure before binding to the SH2 domain, rather it is the interaction between the two that imposes the hairpin loop to the peptide. Furthermore, we find that the peptide adopts a similar structure when bound to both the wild-type Grb2 SH2 domain and a TrpEF1Gly mutant. This suggests that TrpEF1 is not the determining factor for the conformation of the phosphopeptide., (© 2015 The Protein Society.)

Background: Recent failures of clinical trials promoting HDL-elevating therapies have prompted research groups to focus on its functional activity in disease. Endothelial effects of HDL can be measured with in vitro cell assays. The reproducibility and biological relevance of these assays have not been explored both in healthy individuals and those at increased cardiovascular (CV) risk., Methods: HDL dependent nitric oxide (NO) bioavailability, superoxide (SO) production and serum paraoxonase-1 (PON-1) activity were measured in 35 healthy adults (34.37 24-49) and 8 patients (43.56 37-49) suffering from a chronic inflammatory condition (periodontitis-PD). Assay reproducibility was assessed by independent technicians on consecutive days to determine inter and intra analyser variability for each assay. The 35 healthy individuals were further divided into young (n = 16) and middle aged (n = 19) groups and compared with regards to HDL functions. Within-subject biological variation of HDL function was determined in a sub-group of 25 healthy volunteers at intervals of one day and 1 month, and in 8 patients at intervals of one day and 1 week. Power curves were also generated to estimate the number of patients that would be required for HDL functional assays in a cross-over and parallel study design., Results: NO bioavailability was the most reproducible assay in healthy adults (coefficient of variation = 1.72%, 1.92 - intra and inter respectively) and PD patients (CV = 4.4% and 5.5%). All measures demonstrated no statistical difference between young and healthy middle aged population. No single assay demonstrated significant variations over time, indicating that within patient variations are negligible. Our power curves for NO bioavailability and PON-1 activity suggest that low number of patients will be required to detect significant differences in HDL function in a cross over and parallel study design., Conclusion: This study suggests that in vitro HDL functional assays are reliable and can be used to assess HDL functionality in healthy and diseased populations. NO bioavailability was the most reproducible assay, but PON-1 activity remains the most practical for application in clinical trials due to its capacity for scale., (Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.)

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes.

Objective: HDL functionality has been shown to be impaired in inflammatory conditions, including coronary artery disease. The present study aims to determine the impact of low grade and acute inflammation on HDL function and structure., Approach and Results: i) The endothelial protective effects of HDL were compared between 26 periodontal patients and 26 age and sex matched controls by measuring paraoxonase activity in serum and nitric oxide bioavailability and superoxide production in endothelial cells. Paraoxonase activity and nitric oxide bioavailability were reduced, while superoxide production was increased (p<0.01) in periodontal patients compared to controls. ii) HDL function, including cholesterol efflux and vascular cell adhesion molecule-1 expression, was subsequently measured in the periodontal patients following an inflammatory stimulus. There was an acute deterioration in HDL's endothelial protective function, without change in cholesterol efflux, after 24h (p<0.01 for all). These functional changes tracked increases of inflammatory markers and altered HDL composition. Finally, HDL function returned to baseline levels after resolution of inflammation., Conclusion: This study demonstrates that even minor alterations in systemic inflammation can impair the endothelial protective effects of HDL. These functional changes were independent of cholesterol efflux and were associated with remodeling of the HDL proteome. All measures of HDL's endothelial protective functions recovered with resolution of inflammation. These findings suggest that HDL dysfunction may represent a novel mechanism linking inflammation with progression of atheroma., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Pulsed electron paramagnetic resonance (EPR) spectroscopy using microwaves at two frequencies can be employed to measure distances between pairs of paramagnets separated by up to 10 nm. The method, combined with site-directed mutagenesis, has become increasingly popular in structural biology for both its selectivity and capability of providing information not accessible through more standard methods such as nuclear magnetic resonance and X-ray crystallography. Despite these advantages, EPR distance measurements suffer from poor sensitivity. One contributing factor is technical: since 65 MHz typically separates the pump and detection frequencies, they cannot both be located at the center of the pseudo-Lorentzian microwave resonance of a single-mode resonator. To maximize the inversion efficiency, the pump pulse is usually placed at the center of the resonance, while the observer frequency is placed in the wing, with consequent reduction in sensitivity. Here, we consider an alternative configuration: by spacing pump and observer frequencies symmetrically with respect to the microwave resonance and by increasing the quality factor, valuable improvement in the signal-to-noise ratio can be obtained.

Photocatalytic generation of reactive oxygen species (ROS) from quantum dots (QDs) has been widely reported yet quantitative studies of ROS formation and their quantum yields are lacking. This study investigates the generation of ROS by water soluble PEGylated CdSe/ZnS QDs with red emission. PEGylation of QDs is commonly used to confer water solubility and minimise uptake by organs of the reticuloendothelial system; therefore studies of ROS formation are of biomedical relevance. Using non-photolytic visible wavelength excitation, the superoxide anion radical is shown to be the primary ROS species generated with a quantum efficiency of 0.35%. The yield can be significantly enhanced in the presence of the electron donor, nicotinamide adenine dinucleotide (NADH), as demonstrated by oxygen consumption measurements and electron paramagnetic resonance spectroscopy with in situ illumination. Direct production of singlet oxygen is not detectable from the QDs alone. A comparison is made with ROS generation by the same QDs complexed with a sulfonated phthalocyanine which can generate singlet oxygen via Förster resonance energy transfer between the QDs and the phthalocyanine., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Subunit 5 of Saccharomyces cerevisiae cytochrome c oxidase (CcO) is essential for assembly and has two isoforms, 5A and 5B. 5A is expressed under normoxic conditions, whereas 5B is expressed at very low oxygen tensions. As a consequence, COX5A-deleted strains (Δcox5A) have no or only low levels of CcO under normoxic conditions rendering them respiratory deficient. Previous studies have reported that respiratory growth could be restored by combining Δcox5A with mutations of ROX1 that encodes a repressor of COX5B expression. In these mutants, 5B isoenzyme expression level was 30-50% of wild-type (5A isoenzyme) and exhibited a maximum catalytic activity up to 3-fold faster than that of 5A isoenzyme. To investigate the origin of this effect, we constructed a mutant strain in which COX5B replaced COX5A downstream of the COX5A promoter. This strain expressed wild-type levels of the 5B isoenzyme, without the complication of additional effects caused by mutation of ROX1. When produced this way, the isoenzymes displayed no significant differences in their maximum catalytic activities or in their affinities for oxygen or cytochrome c. Hence the elevated activity of the 5B isoenzyme in the rox1 mutant is not caused simply by exchange of isoforms and must arise from an additional effect that remains to be resolved.

The viability of dilute magnetic semiconductors in applications is linked to the strength of the magnetic couplings, and room temperature operation is still elusive in standard inorganic systems. Molecular semiconductors are emerging as an alternative due to their long spin-relaxation times and ease of processing, but, with the notable exception of vanadium-tetracyanoethylene, magnetic transition temperatures remain well below the boiling point of liquid nitrogen. Here we show that thin films and powders of the molecular semiconductor cobalt phthalocyanine exhibit strong antiferromagnetic coupling, with an exchange energy reaching 100 K. This interaction is up to two orders of magnitude larger than in related phthalocyanines and can be obtained on flexible plastic substrates, under conditions compatible with routine organic electronic device fabrication. Ab initio calculations show that coupling is achieved via superexchange between the singly occupied a1g () orbitals. By reaching the key milestone of magnetic coupling above 77 K, these results establish quantum spin chains as a potentially useable feature of molecular films.

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG is one of three maturase enzymes involved in [FeFe]-hydrogenase H-cluster assembly. It catalyzes L-tyrosine cleavage to yield the H-cluster cyanide and carbon monoxide ligands as well as p-cresol. Clostridium acetobutylicum HydG contains the conserved CX3CX2C motif coordinating the AdoMet binding [4Fe-4S] cluster and a C-terminal CX2CX22C motif proposed to coordinate a second [4Fe-4S] cluster. To improve our understanding of the roles of each of these iron-sulfur clusters in catalysis, we have generated HydG variants lacking either the N- or C-terminal cluster and examined these using spectroscopic and kinetic methods. We have used iron analyses, UV-visible spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy of an N-terminal C96/100/103A triple HydG mutant that cannot coordinate the radical AdoMet cluster to unambiguously show that the C-terminal cysteine motif coordinates an auxiliary [4Fe-4S] cluster. Spectroscopic comparison with a C-terminally truncated HydG (ΔCTD) harboring only the N-terminal cluster demonstrates that both clusters have similar UV-visible and EPR spectral properties, but that AdoMet binding and cleavage occur only at the N-terminal radical AdoMet cluster. To elucidate which steps in the catalytic cycle of HydG require the auxiliary [4Fe-4S] cluster, we compared the Michaelis-Menten constants for AdoMet and L-tyrosine for reconstituted wild-type, C386S, and ΔCTD HydG and demonstrate that these C-terminal modifications do not affect the affinity for AdoMet but that the affinity for L-tyrosine is drastically reduced compared to that of wild-type HydG. Further detailed kinetic characterization of these HydG mutants demonstrates that the C-terminal cluster and residues are not essential for L-tyrosine cleavage to p-cresol but are necessary for conversion of a tyrosine-derived intermediate to cyanide and CO.

Organic semiconductors are studied intensively for applications in electronics and optics, and even spin-based information technology, or spintronics. Fundamental quantities in spintronics are the population relaxation time (T1) and the phase memory time (T2): T1 measures the lifetime of a classical bit, in this case embodied by a spin oriented either parallel or antiparallel to an external magnetic field, and T2 measures the corresponding lifetime of a quantum bit, encoded in the phase of the quantum state. Here we establish that these times are surprisingly long for a common, low-cost and chemically modifiable organic semiconductor, the blue pigment copper phthalocyanine, in easily processed thin-film form of the type used for device fabrication. At 5 K, a temperature reachable using inexpensive closed-cycle refrigerators, T1 and T2 are respectively 59 ms and 2.6 μs, and at 80 K, which is just above the boiling point of liquid nitrogen, they are respectively 10 μs and 1 μs, demonstrating that the performance of thin-film copper phthalocyanine is superior to that of single-molecule magnets over the same temperature range. T2 is more than two orders of magnitude greater than the duration of the spin manipulation pulses, which suggests that copper phthalocyanine holds promise for quantum information processing, and the long T1 indicates possibilities for medium-term storage of classical bits in all-organic devices on plastic substrates.

Although the major light harvesting complexes of diatoms, called FCPs (fucoxanthin chlorophyll a/c binding proteins), are related to the cab proteins of higher plants, the structures of these light harvesting protein complexes are much less characterized. Here, a structural/functional model for the "core" of FCP, based on the sequence homology with LHCII, in which two fucoxanthins replace the central luteins and act as quenchers of the Chl a triplet states, is proposed. Combining the information obtained by time-resolved EPR spectroscopy on the triplet states populated under illumination, with quantum mechanical calculations, we discuss the chlorophyll triplet quenching in terms of the geometry of the chlorophyll-carotenoid pairs participating to the process. The results show that local structural rearrangements occur in FCP, with respect to LHCII, in the photoprotective site., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The photoexcitation of functionalized fullerenes to their paramagnetic triplet electronic state can be studied by pulsed electron paramagnetic resonance (EPR) spectroscopy, whereas the interactions of this state with the surrounding nuclear spins can be observed by a related technique: electron nuclear double resonance (ENDOR). In this study, we present EPR and ENDOR studies on a functionalized exohedral fullerene system, dimethyl[9-hydro (C60-Ih)[5,6]fulleren-1(9H)-yl]phosphonate (DMHFP), where the triplet electron spin has been used to hyperpolarize, couple and measure two nuclear spins. We go on to discuss the extension of these methods to study a new class of endohedral fullerenes filled with small molecules, such as H₂@C₆₀, and we relate the results to density functional calculations.

The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.

Pulsed magnetic resonance allows the quantum state of electronic and nuclear spins to be controlled on the timescale of nanoseconds and microseconds respectively. The time required to flip dilute spins is orders of magnitude shorter than their coherence times, leading to several schemes for quantum information processing with spin qubits. Instead, we investigate 'hybrid nuclear-electronic' qubits consisting of near 50:50 superpositions of the electronic and nuclear spin states. Using bismuth-doped silicon, we demonstrate quantum control over these states in 32 ns, which is orders of magnitude faster than previous experiments using pure nuclear states. The coherence times of up to 4 ms are five orders of magnitude longer than the manipulation times, and are limited only by naturally occurring (29)Si nuclear spin impurities. We find a quantitative agreement between our experiments and an analytical theory for the resonance positions, as well as their relative intensities and Rabi oscillation frequencies. These results bring spins in a solid material a step closer to research on ion-trap qubits.

Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.

A major obstacle to the efficient production of antibody conjugates for therapy and diagnosis is the non-ideal performance of commonly used chemical methods for the attachment of effector-molecules to the antibody of interest. Here we demonstrate that this limitation can be simply addressed using 3,4-substituted maleimides to bridge and thus functionalize disulfide bonds to generate homogeneous antibody conjugates. This one-step conjugation reaction is fast, site-specific, quantitative and generates products with full binding activity, good plasma stability and the desired functional properties. Furthermore, the rigid nature of this modification by disulfide bridging enables the successful detection of antigen with a spin labeled antibody fragment by continuous-wave electron paramagnetic resonance (cw-EPR), which we report here for the first time. Antigen detection is concentration dependent, observable in human blood and allows the discrimination of fragments with different binding affinity. We envisage broad potential for antibody based in-solution diagnostic methods by EPR or 'spinostics'.

Nanostructure and molecular orientation play a crucial role in determining the functionality of organic thin films. In practical devices, such as organic solar cells consisting of donor-acceptor mixtures, crystallinity is poor and these qualities cannot be readily determined by conventional diffraction techniques, while common microscopy only reveals surface morphology. Using a simple nondestructive technique, namely, continuous-wave electron paramagnetic resonance spectroscopy, which exploits the well-understood angular dependence of the g-factor and hyperfine tensors, we show that in the solar cell blend of C(60) and copper phthalocyanine (CuPc)-for which X-ray diffraction gives no information-the CuPc, and by implication the C(60), molecules form nanoclusters, with the planes of the CuPc molecules oriented perpendicular to the film surface. This information demonstrates that the current nanostructure in CuPc:C(60) solar cells is far from optimal and suggests that their efficiency could be considerably increased by alternative film growth algorithms.

Carotenoid molecules are essential for the life of photosynthetic organisms in that they protect the cell from the photo-oxidative damage induced by light-stress conditions. One of the photo-protective mechanisms involves triplet-triplet energy transfer from the chlorophyll molecules to the carotenoids: a process that is strongly dependent on the electronic properties of the triplet states involved. Here, we obtain a clear description of the triplet state of lutein in LHCII from higher plants for the first time by density functional theory (DFT) calculations. DFT predictions have been validated by comparison with hyperfine couplings obtained with pulsed-ENDOR spectroscopy. Knowledge of the spin density distribution, the frontier orbitals and orbital excitations forms a basis for discussing the requirements for an efficient triplet-triplet energy transfer. The results obtained for the lutein in LHCII are compared with those of the highly-substituted carotenoid peridinin in PCP from Amphidinium carterae [Di Valentin et al., Biochim. Biophys. Acta, 2008, 1777, 295-307]. The presence of substituents in the peridinin molecule does not alter significantly the triplet state electronic structure compared to lutein. Despite the unusual spectroscopic behaviour of the peridinin excited singlet state, lutein and peridinin have similar triplet state properties. In both molecules the unpaired spins are delocalized uniformly over the whole π-conjugated system in an alternating even-odd pattern.

We present pulsed electron-nuclear double resonance (ENDOR) experiments which enable us to characterize the coupling between bismuth donor spin-qubits in Si and the surrounding spin-bath of (29)Si impurities which provides the dominant decoherence mechanism (nuclear spin diffusion) at low temperatures (< 16 K). Decoupling from the spin-bath is predicted and cluster correlation expansion simulations show near-complete suppression of spin diffusion, at optimal working points. The suppression takes the form of sharply peaked divergences of the spin diffusion coherence time, in contrast with previously identified broader regions of insensitivity to classical fluctuations. ENDOR data shows anisotropic contributions are comparatively weak, so the form of the divergences is independent of crystal orientation.

UvrB has a central role in the highly conserved UvrABC pathway functioning not only as a damage recognition element but also as an essential component of the lesion tracking machinery. While it has been recently confirmed that the tracking assembly comprises a UvrA2B2 heterotetramer, the configurations of the damage engagement and UvrB-DNA handover complexes remain obscure. Here, we present the first crystal structure of a UvrB dimer whose biological significance has been verified using both chemical cross-linking and electron paramagnetic resonance spectroscopy. We demonstrate that this dimeric species stably associates with UvrA and forms a UvrA2B2-DNA complex. Our studies also illustrate how signals are transduced between the ATP and DNA binding sites to generate the helicase activity pivotal to handover and formation of the UvrB2-DNA complex, providing key insights into the configurations of these important repair intermediates.

Iron-catalyzed generation of free radicals leads to molecular damage in vivo, and has been proposed to contribute to organismal ageing. Here we investigate the role of free iron in ageing in the nematode Caenorhabditis elegans. Media supplementation with Fe(III) increased free iron levels in vivo, as detected by continuous-wave electron paramagnetic resonance spectroscopy and elevated expression of the iron-sensitive reporter transgene pftn-1::gfp. Increased free iron levels caused elevated levels of protein oxidation and hypersensitivity to tert-butyl hydroperoxide (t-BOOH) given 9 mM Fe(III) or greater, but 15 mM Fe(III) or greater was required to reduce lifespan. Treatment with either an iron chelator (deferoxamine) or over-expression of ftn-1, encoding the iron sequestering protein ferritin, increased resistance to t-BOOH and, in the latter case, reduced protein oxidation, but did not increase lifespan. Expression of ftn-1 is greatly increased in long-lived daf-2 insulin/IGF-1 receptor mutants. In this context, deletion of ftn-1 decreased t-BOOH resistance, but enhanced both daf-2 mutant longevity and constitutive dauer larva formation, suggesting an effect of ferritin on signaling. These results show that high levels of iron can increase molecular damage and reduce lifespan, but overall suggest that iron levels within the normal physiological range do not promote ageing in C. elegans., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by Förster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.

Type 1 pili are the archetypal representative of a widespread class of adhesive multisubunit fibres in Gram-negative bacteria. During pilus assembly, subunits dock as chaperone-bound complexes to an usher, which catalyses their polymerization and mediates pilus translocation across the outer membrane. Here we report the crystal structure of the full-length FimD usher bound to the FimC-FimH chaperone-adhesin complex and that of the unbound form of the FimD translocation domain. The FimD-FimC-FimH structure shows FimH inserted inside the FimD 24-stranded β-barrel translocation channel. FimC-FimH is held in place through interactions with the two carboxy-terminal periplasmic domains of FimD, a binding mode confirmed in solution by electron paramagnetic resonance spectroscopy. To accommodate FimH, the usher plug domain is displaced from the barrel lumen to the periplasm, concomitant with a marked conformational change in the β-barrel. The amino-terminal domain of FimD is observed in an ideal position to catalyse incorporation of a newly recruited chaperone-subunit complex. The FimD-FimC-FimH structure provides unique insights into the pilus subunit incorporation cycle, and captures the first view of a protein transporter in the act of secreting its cognate substrate.

The neutral and anionic semiquinone radicals of the flavin adenine dinucleotide (FAD) cofactor noncovalently bound in glucose oxidase from A. niger are examined with the aid of QM/MM molecular modeling methods, enabling complete inclusion of the protein environment. Recently, the electron paramagnetic resonance (EPR) characteristics, the anisotropic g tensor and all the significant hyperfine couplings, of these flavoprotein radicals were determined at high resolution (J. Phys. Chem. B 2008, 112, 3568). A striking difference between the neutral and anionic radical forms was found to be a shift in the g(y) principal value. Within the QM/MM framework, geometry optimization and molecular dynamics simulations are combined with EPR property calculations, employing a recent implementation by some of the authors in the CP2K software package. In this way, spectroscopic characteristics are computed on the fly during the MD simulations of the solvated protein structure, mimicking as best as possible the experimental conditions. The general agreement between calculated and experimental EPR properties is satisfactory and on par with those calculated with other codes (Gaussian 03, ORCA). The protonation state of two histidines (His559 and His516) at the catalytic site of this flavoprotein is found to have a remarkable influence on the isotropic hyperfine coupling of one of the methyl groups on the neutral FAD radical, which is consistent with experimental findings in other flavoproteins (J. Biol. Chem. 2007, 282, 4738). Furthermore, the shift in the g(y) principal values between the neutral and anionic radicals is well reproduced by QM/MM simulations. Incorporation of at least the nearest protein environment of the cofactor radicals proves to be vital for a correct reproduction, indicating that this shift is a global feature of the protein rather than a local one. In addition, QM/MM techniques are used to make a prediction of relative angles between important spectroscopic principal directions, which are not readily determined by conventional EPR experiments. Significantly, the directions of the g(x) and the g(y) components of the g-tensor that lie in the plane of the isoalloxazine moiety are rotated by approximately 59° between the neutral and the anionic radicals.

A prerequisite for exploiting spins for quantum data storage and processing is long spin coherence times. Phosphorus dopants in silicon (Si:P) have been favoured as hosts for such spins because of measured electron spin coherence times (T2) longer than any other electron spin in the solid state: 14 ms at 7 K with isotopically purified silicon. Heavier impurities such as bismuth in silicon (Si:Bi) could be used in conjunction with Si:P for quantum information proposals that require two separately addressable spin species. However, the question of whether the incorporation of the much less soluble Bi into Si leads to defect species that destroy coherence has not been addressed. Here we show that schemes involving Si:Bi are indeed feasible as the electron spin coherence time T2 is at least as long as for Si:P with non-isotopically purified silicon. We polarized the Si:Bi electrons and hyperpolarized the I=9/2 nuclear spin of (209)Bi, manipulating both with pulsed magnetic resonance. The larger nuclear spin means that a Si:Bi dopant provides a 20-dimensional Hilbert space rather than the four-dimensional Hilbert space of an I=1/2 Si:P dopant.

RNA polymerases (RNAP) carry out transcription, the first step in the highly regulated process of gene expression. RNAPs are complex multisubunit enzymes, which undergo extensive structural rearrangements during the transcription cycle (initiation-elongation-termination). They accommodate interactions with the nucleic acid scaffold of transcription complexes (template DNA, DNA/RNA hybrid, and nascent RNA) and interact with a plethora of transcription factors. Here we focused on the RNAP-F/E subcomplex, which forms a stable heterodimer that binds the nascent RNA and thereby stimulates the processivity of elongation complexes. We used the pulsed-EPR method DEER and fluorescence spectroscopy to probe for conformational changes within the F/E dimer. Our results demonstrate that, upon binding of RNA, F/E remains in a stable conformation, which suggests that it serves as a structurally rigid guiding rail for the growing RNA chain during transcription.

A series of butadiyne-linked zinc porphyrin oligomers, with one, two, three, and four porphyrin units and lengths of up to 75 A, have been spin-labeled at both ends with stable nitroxide TEMPO radicals. The pulsed EPR technique of double electron electron resonance (DEER) was used to probe the distribution of intramolecular end-to-end distances, under a range of conditions. DEER measurements were carried out at 50 K in two types of dilute solution glasses: deutero-toluene (with 10% deutero-pyridine) and deutero-o-terphenyl (with 5% 4-benzyl pyridine). The complexes of the porphyrin oligomers with monodentate ligands (pyridine or 4-benzyl pyridine) principally adopt linear conformations. Nonlinear conformations are less populated in the lower glass-transition temperature solvent. When the oligomers bind star-shaped multidentate ligands, they are forced to bend into nonlinear geometries, and the experimental end-to-end distances for these complexes match those from molecular mechanics calculations. Our results show that porphyrin-based molecular wires are shape-persistent, and yet that their shapes can deformed by binding to multivalent ligands. Self-assembled ladder-shaped 2:2 complexes were also investigated to illustrate the scope of DEER measurements for providing structural information on synthetic noncovalent nanostructures.

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), and (-)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O(2)(-)) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O(2)(-) oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O(2)(-) oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.

All pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases contain an unusual disulfide ring formed between adjacent cysteine residues. A mutant enzyme that is lacking this structure was generated by replacing Cys105 and Cys106 with Ala in quinoprotein ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa ATCC17933. Heterologously expressed quinoprotein ethanol dehydrogenase in which Cys-105 and Cys-106 have been replaced by Ala (Cys105Ala/Cys106Ala apo-QEDH) was successfully converted to enzymatic active holo-enzyme by incorporation of its cofactor PQQ in the presence of Ca(2+). The enzymatic activity of the mutant enzyme in the artificial dye test with N-methylphenazonium methyl sulfate (PMS) and 2,6-dichlorophenol indophenol (DCPIP) at pH 9 did not depend on an activating amine which is essential for wild type activity under these conditions. The mutant enzyme showed increased Michaelis constants for primary alcohols, while the affinity for the secondary alcohol 2-propanol was unaltered. Surprisingly, for all substrates tested the specific activity of the mutant enzyme in the artificial dye test was higher than that found for wild type QEDH. On the contrary, in the ferricyanide test with the natural electron acceptor cytochrome c(550) the activity of mutant Cys105Ala/Cys106Ala was 15-fold lower than that of wild type QEDH. We demonstrate for the first time unambiguously that the unusual disulfide ring is essential for efficient electron transfer at pH 7 from QEDH to its natural electron acceptor cytochrome c(550).

Free radical generation as a result of oxidation reactions of rosmarinic acid (RA), a caffeic acid ester with 3,4-dihydroxyphenyllactate, was investigated by electron paramagnetic resonance (EPR) spectroscopy using a variety of oxidation conditions. Limitations and possibilities of using the various methodologies to obtain information about the reaction chemistry of polyphenols are discussed. Three different spectra were detected dependent on the pH and oxidizing agent. Feasible structures for the observed radicals were tested by density functional theory (DFT) calculations and the results indicate that oxidation reactions can occur at both of the catechol groups.

The catalytic cofactor thiamine diphosphate is found in many enzymes of central metabolism and is essential in all extant forms of life. We demonstrate the presence of an oxygen-dependent free radical in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The radical was sufficiently long-lived to be trapped by freezing in liquid nitrogen, and its electronic structure was investigated by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR). Taken together, the spectroscopic results revealed a delocalized pi radical on the enamine-thiazolium intermediate within the enzyme active site. The radical is generated as an intermediate during substrate turnover by a side reaction with molecular oxygen, resulting in the continuous production of reactive oxygen species under aerobic conditions. This off-pathway reaction may account for metabolic dysfunction associated with several neurodegenerative diseases. The possibility that the on-pathway reaction may proceed via a radical mechanism is discussed.

It was recently suggested that partially reduced monoamine oxidase (MAO) A contains an equilibrium mixture of an anionic flavin radical and a tyrosyl radical (Rigby, S. E.; et al. J. Biol. Chem. 2005, 280, 4627-4632). These observations formed the basis for a revised radical mechanism for MAO. In contrast, an earlier study of MAO B only found evidence for an anionic flavin radical (DeRose, V. J.; et al. Biochemistry 1996, 35, 11085-11091). To resolve the discrepancy, we have performed continuous-wave electron paramagnetic resonance at 94 GHz (W-band) on the radical form of MAO A. A comparison with d-amino acid oxidase (DAAO) demonstrates that both enzymes only contain anionic flavin radicals. Pulsed electron-nuclear double resonance spectra of the two enzymes recorded at 9 GHz (X-band) reveal distinct hyperfine coupling patterns for the two flavins. Density functional theory calculations show that these differences can be understood in terms of the difference at C8alpha of the isoalloxazine ring. DAAO contains a noncovalently bound flavin whereas MAO A contains a flavin covalently bound to a cysteinyl residue at C8alpha. The similar electronic structures and hydrophobic environments of MAO and DAAO, and the similar structural motifs of their substrates suggest that a direct hydride transfer catalytic mechanism established for DAAO (Umhau, S.; et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12463-12468) should be considered for MAO.

Binding of methanol to the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa has been studied by pulsed electron-nuclear double resonance at 9 GHz. Shifts in the hyperfine couplings of the pyrroloquinoline quinone radical provide direct evidence for a change in the environment of the cofactor when substrate is present. By performing experiments with deuteriated methanol, we confirmed that methanol was the cause of the effect. Density functional theory calculations show that these shifts can be understood if a water molecule, which is often found in x-ray structures of the active site of quinoprotein alcohol dehydrogenases, is displaced by the substrate. The difference between the binding of water and methanol is that the water molecule forms a hydrogen bond to O5 of pyrroloquinoline quinone, which the methanol, by virtue of its methyl group, does not. The results support the proposal that aspartate rather than glutamate is the catalytically active base for a hydride transfer mechanism in quinoprotein alcohol dehydrogenases.

Quinoprotein alcohol dehydrogenases use the pyrroloquinoline quinone (PQQ) cofactor to catalyze the oxidation of alcohols. The catalytic cycle is thought to involve a hydride transfer from the alcohol to the oxidized PQQ, resulting in the generation of aldehyde and reduced PQQ. Reoxidation of the cofactor by cytochrome proceeds in two sequential steps via the PQQ radical. We have used a combination of electron nuclear double resonance and density functional theory to show that the PQQ radical is not protonated at either O-4 or O-5, a result that is at variance with the general presumption of a singly protonated radical. The quantum mechanical calculations also show that reduced PQQ is unlikely to be protonated at O-5; rather, it is either singly protonated at O-4 or not protonated at either O-4 or O-5, a result that also challenges the common assumption of a reduced PQQ protonated at both O-4 and O-5. The reaction cycle of PQQ-dependent alcohol dehydrogenases is revised in light of these findings.

Pulsed electron-electron double resonance (ELDOR) has been used to obtain structural information from a FAD-dependent sulfhydryl oxidase, Augmenter of Liver Regeneration (ALR). ALR is a homodimer with each subunit containing a noncovalently bound FAD cofactor. Both FADs may be converted into the blue neutral radical form by aerobic treatment with DTT. From three-pulse and four-pulse ELDOR experiments, a distance of 26.1 +/- 0.8 A could be determined between the FAD cofactors in human ALR. Taking into account the electron spin density distribution in a neutral flavin radical obtained from density functional theory calculations, a distance of 26.9 A could be estimated for the separation of the spin centers in the X-ray structure of rat ALR. The good agreement confirms that rat ALR may be used as a model for mechanistic discussions of human ALR. The experiments also demonstrate that neutral flavin radicals have the appropriate properties to be used as intrinsic spin labels for distance determinations in proteins.

A high-microwave-frequency/high-magnetic-field pulsed electron-nuclear double resonance (ENDOR) study performed at 94 GHz on the flavin semiquinone cofactor of Xenopus laevis (6-4) photolyase in its neutral radical state is presented. Although the principal values of the flavin radical's g-matrix are not fully resolved in the 94-GHz EPR spectrum in a nonoriented sample, the orientation of the principal axes of g is obtained by exploiting the orientation selection of the proton ENDOR signals from the methyl protons at C-8alpha and the deuteron ENDOR signals from D-5 in an enzyme sample in deuterated buffer. This procedure for assigning the orientation of g relative to the molecular frame makes use of commercially available ENDOR instrumentation without the necessity to perform single-crystal studies., (Copyright 2005 John Wiley & Sons, Ltd)

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

Pyrroloquinoline quinone (2,7,9-tricarboxypyrroloquinoline quinone, PQQ) is one of several quinone cofactors that is utilized in a class of dehydrogenases known as quinoproteins. In this contribution, we have used continuous-wave high-field/high-frequency electron paramagnetic resonance (EPR) at 94 GHz (W-band) to study substrate binding in ethanol dehydrogenase (QEDH) from Pseudomonas aeruginosa, taking advantage of the fact that the enzyme is isolated with a substantial proportion of the PQQ cofactor in the paramagnetic semiquinone form. In the substrate-free enzyme, the principal values of the g-tensor, obtained by spectral simulation are: gx = 2.00585(2), gy = 2.00518(2), and gz = 2.00212(2), giving giso = 2.00438(2). All three principal values of the g-tensor decrease when ethanol is bound to the protein: gx = 2.00574(2), gy = 2.00511(2), and gz = 2.00207(2), giving giso = 2.00431(2). The results represent the first direct evidence for the tight binding of an alcohol to a PQQ-dependent alcohol dehydrogenase and show that ethanol also binds to the enzyme even when the PQQ cofactor is in the semiquinone form. The decrease in g is consistent with an increase in polarity in the immediate vicinity of the PQQ cofactor and probably reflects a changed geometry of the PQQ-Ca2+ complex when ethanol binds.

An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.

The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.

The binding pocket of the pyrroloquinoline quinone (PQQ) cofactor in quinoprotein alcohol dehydrogenases contains a characteristic disulphide ring formed by two adjacent cysteine residues. To analyse the function of this unusual structural motif we have investigated the wild-type and a double cysteine:alanine mutant of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa by electron paramagnetic resonance (EPR) spectroscopy. Thus, we have obtained the principal values for the full rhombic g-tensor of the PQQ semiquinone radical by high-field (94 GHz) EPR necessary for a discrimination of radical species in dehydrogenases containing PQQ together with other redox-active cofactors. Our results show that the characteristic disulphide ring is no prerequisite for the formation of the functionally important semiquinone form of PQQ.

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

In the flavin mononucleotide-binding LOV1 domain of the Phot1-receptor from Chlamydomonas reinhardtii the photoreactive cysteine C57 has been replaced by methionine. Photoexcitation of this C57M mutant yields a metastable photoproduct (C57M-415) that thermally decomposes into a stable paramagnetic species (C57M-675) with extremely red-shifted absorption in the visible range. In this contribution, we describe the characterization of this radical by multi-frequency electron paramagnetic resonance and electron-nuclear double resonance. The main features of the spectra identify the paramagnetic species as a flavin neutral radical. However, detailed analysis shows that the isoalloxazine moiety of the flavin is alkyl substituted at N(5), rather than protonated as is usually the case. The implication of these observations on the likely mechanism of photoproduct generation in wild-type LOV domains is discussed.