Water-Soluble Chlorophyll Proteins (WSCPs) from Brassicaceae are non-photosynthetic proteins which tetramerize upon binding four chlorophyll (Chl) molecules. The bound Chls are highly photostable, despite the lack of bound carotenoids known, in Chl-containing photosynthetic proteins, to act as singlet oxygen and Chl triplet (3Chl) quenchers. Although the physiological function of WSCPs is still unclear, it is likely to be related to their biochemical stability and their resistance to photodegradation. To get insight into the origin of this photostability, the properties of the 3Chl generated in WSCPs upon illumination were investigated. We found that, unlike the excited singlet states, which are excitonic states, the triplet state is localized on a single Chl molecule. Moreover, the lifetime of the 3Chl generated in WSCPs is comparable to that observed in other Chl-containing systems and is reduced in presence of oxygen. In contrast to previous observations, we found that WSCP actually photosensitizes singlet oxygen with an efficiency comparable to that of Chl in organic solvent. We demonstrated that the observed resistance to photooxidation depends on the conformation of the phytyl moieties, which in WSCP are interposed between the rings of Chl dimers, hindering the access of singlet oxygen to the oxidizable sites of the pigments.

This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron double resonance (PELDOR/DEER) to determine the interspin distance between a photoexcited porphyrin triplet state (S = 1) and a nitroxide spin label chemically incorporated into a small helical peptide. The PELDOR trace shows deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair, providing an accurate distance measurement. This new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the sensitivity acquired from the spin polarization of the photoexcited triplet state spectrum.

[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe-4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe-4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron-sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx(46-53)HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.

Wear-out and breakdown of polymers used as insulating materials are closely related to the dissipation of electrostatic energy in space charge regions. A fraction of this energy is radiated off by electroluminescence which can be considered as a pre-breakdown warning and provides a probe to investigate the mechanisms leading to structural degradation. Electroluminescence emission from two aromatic polyesters is studied - poly(ethylene terephthalate) and poly(ethylene 2,6-naphthalate). We investigate the excitation processes of the luminescent centres, the nature of the centres, and the relationship of the emission features to electrical ageing. Sample electroluminescence is excited by dc and ac voltages under a uniform field configuration. The discussion relies on the analysis of the charge transport process in the luminescent regime, the acquisition of electroluminescence and photoluminescence spectra, and the investigation of the field effect on photoluminescence.

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin-labeling method based on the photoexcited triplet state (S=1). Four-pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N-terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8-8 nm. The PELDOR traces provide accurate distance measurements for all the ruler series, showing deep envelope modulations at frequencies varying in a progressive way according to the increasing distance between the spin labels. The upper limit is evaluated and found to be around 8 nm. The PELDOR-derived distances are in excellent agreement with theoretical predictions. We demonstrate that high sensitivity is acquired using the triplet state as a spin label by comparison with Cu(II)-porphyrin analogues. The new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the properties of the porphyrin triplet state.

The rheological properties of gas cell walls in bread doughs are considered to be important in relation to their stability and gas retention during proof and baking. Large deformation rheological properties of gas cell walls were measured using biaxial extension for a number of doughs of varying breadmaking quality at constant strain rate and elevated temperatures of 25–60°C. Strain hardening and failure strain of cell walls both decreased with temperature, with cell walls in good breadmaking doughs remaining stable and retaining their strain hardening properties at higher temperatures (60°C), while the cell walls of poor breadmaking doughs became unstable at lower temperatures (45–50°C) and had lower strain hardening. Strain hardening measured at 50°C gave good correlations with baking volume, with the best correlations achieved between rheological measurements and baking tests that used similar mixing conditions. As predicted by the Considere failure criterion, a strain hardening value of 1 defines a region below which gas cell walls become unstable, and discriminates well between the baking quality of a range of commercial flour blends of varying quality. This indicates that the stability of gas cell walls during baking is strongly related to strain hardening properties, and that extensional rheological measurements can be used as indicators of baking quality.

The nature of the photoexcited triplet state of free-base 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (H2TPPS4−) has been investigated by advanced Electron Paramagnetic Resonance (EPR) techniques combined with quantum chemical calculations. The zero-field splitting (ZFS) parameters, D and E, the orientation of the transition dipole moment in the ZFS tensor frame, and the proton hyperfine couplings have been determined by magnetophotoselection-EPR and pulse electron-nuclear double resonance spectroscopy. Both time-resolved and pulse experiments exploit the electron spin polarization of the photoexcited triplet state. Comparison of the magnetic observables with computational results, including CASSCF calculations of the ZFS interaction tensor, provides an accurate picture of the triplet-state electronic structure. The theoretical investigation has been integrated with a systematic analysis on the parent free-base porphyrin molecule to assess the effect of the sulfonatophenyl substituents on the magnetic tensors. Additionally, the magnetophotoselection effects are discussed in terms of tautomerization in the excited singlet state of H2TPPS4−.

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