Detailed investigation of the binding abilities of the heterodomain of a multiHis cyclopeptide toward Cu(II) ions.

Cyclopeptides hold significant relevance in various fields of science and medicine, due to their unique structural properties and diverse biological activities. Cyclic peptides, characterized by intrinsically higher conformational order, exhibit remarkable stability and resistance to proteolytic degradation, making them attractive candidates for developing targeted drug delivery systems. The aim of this work is to elucidate the unique coordination properties of the multi-His cyclic peptide with c(HDHKHPHHKHHP) sequence (HDCP - heterodomain cyclopeptide). This peptide, indeed, is able to form homo- and hetero-dinuclear complexes in a wide pH range, being thus a good chelator for Cu(II) ions. Herein, we present the results of a combined study, involving potentiometric, spectroscopic (UV-Vis, CD, and EPR), and computational investigations, on its coordination properties. To better understand the interaction pattern with Cu(II) metal ions, two other peptides, each one bearing only one of the two binding domains of HDCP are also considered in this study: c(HDHKHPGGKGGP) = CP1, c(GKGGKPHHKHHP) = CP2, which share sequence fragments of HDCP and allow separate investigations of its coordination domains.

Determining and controlling conformational information from orientationally selective light-induced triplet-triplet electron resonance spectroscopy for a set of bis-porphyrin rulers.

We recently reported a new technique, light-induced triplet-triplet electron resonance (LITTER) spectroscopy, which allows quantification of the dipolar interaction between the photogenerated triplet states of two chromophores. Here we carry out a systematic LITTER study, considering orientation selection by the detection pulses, of a series of bis-porphyrin model peptides with different porphyrin-porphyrin distances and relative orientations. Orientation-dependent analysis of the dipolar datasets yields conformational information of the molecules in frozen solution which is in good agreement with density functional theory predictions. Additionally, a fast partial orientational-averaging treatment produces distance distributions with minimized orientational artefacts. Finally, by direct comparison of LITTER data to double electron-electron resonance (DEER) measured on a system with Cu(II) coordinated into the porphyrins, we demonstrate the advantages of the LITTER technique over the standard DEER methodology. This is due to the remarkable spectroscopic properties of the photogenerated porphyrin triplet state. This work sets the basis for the use of LITTER in structural investigations of unmodified complex biological macromolecules, which could be combined with Förster resonance energy transfer and microscopy inside cells.

Control of excitation selectivity in pulse EPR on spin-correlated radical pairs with shaped pulses.

Spin-correlated radical pairs generated by photoinduced electron transfer are characterised by a distinctive spin polarisation and a unique behaviour in pulse electron paramagnetic resonance (EPR) spectroscopy. Under non-selective excitation, an out-of-phase echo signal modulated by the dipolar and exchange coupling interactions characterising the radical pair is observed and allows extraction of geometric information in the two-pulse out-of-phase electron spin echo envelope modulation (ESEEM) experiment. The investigation of the role of spin-correlated radical pairs in a variety of biological processes and in the fundamental mechanisms underlying device function in optoelectronics, as well as their potential use in quantum information science, relies on the ability to precisely address and manipulate the spins using microwave pulses. Here, we explore the use of shaped pulses for controlled narrowband selective and broadband non-selective excitation of spin-correlated radical pairs in two model donor-bridge-acceptor triads, characterised by different spectral widths, at X- and Q-band frequencies. We demonstrate selective excitation with close to rectangular excitation profiles using BURP (band-selective, uniform response, pure-phase) pulses and complete non-selective excitation of both spins of the radical pair using frequency-swept chirp pulses. The use of frequency-swept pulses in out-of-phase ESEEM experiments enables increased modulation depths and, combined with echo transient detection and Fourier transformation, correlation of the dipolar frequencies with the EPR spectrum and therefore the potential to extract additional information on the donor-acceptor pair geometry.

Direct Comparison between Förster Resonance Energy Transfer and Light-Induced Triplet-Triplet Electron Resonance Spectroscopy.

To carry out reliable and comprehensive structural investigations, the exploitation of different complementary techniques is required. Here, we report that dual triplet-spin/fluorescent labels enable the first parallel distance measurements by electron spin resonance (ESR) and Förster resonance energy transfer (FRET) on exactly the same molecules with orthogonal chromophores, allowing for direct comparison. An improved light-induced triplet-triplet electron resonance method with 2-color excitation is used, improving the signal-to-noise ratio of the data and yielding a distance distribution that provides greater insight than the single distance resulting from FRET.

The electronic structure and dynamics of the excited triplet state of octaethylaluminum(III)-porphyrin investigated with advanced EPR methods.

The photoexcited triplet state of octaethylaluminum(III)-porphyrin (AlOEP) was investigated by time-resolved Electron Paramagnetic Resonance, Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation in an organic glass at 10 and 80 K. This main group element porphyrin is unusual because the metal has a small ionic radius and is six-coordinate with axial covalent and coordination bonds. It is not known whether triplet state dynamics influence its magnetic resonance properties as has been observed for some transition metal porphyrins. Together with density functional theory modelling, the magnetic resonance data of AlOEP allow the temperature dependence of the zero-field splitting (ZFS) parameters, D and E, and the proton AZZ hyperfine coupling (hfc) tensor components of the methine protons, in the zero-field splitting frame to be determined. The results provide evidence that the ZFS, hfc and spin-lattice relaxation are indeed influenced by the presence of a dynamic process that is discussed in terms of Jahn-Teller dynamic effects. Thus, these effects should be taken into account when interpreting EPR data from larger complexes containing AlOEP.

Insights into the phototautomerism of free-base 5, 10, 15, 20-tetrakis(4-sulfonatophenyl) porphyrin.

Phototautomerism in the excited states of free-base 5, 10, 15, 20-tetrakis(4-sulfonatophenyl) porphyrin (H2TPPS4-) has been investigated combining, for the first time, advanced Electron Paramagnetic Resonance (EPR) with fluorescence and Raman spectroscopy. Triplet EPR spectroscopy, performed in protic and deuterated solvents and in the presence of photoselection, confirms the occurrence of phototautomerization and additionally suggests the formation of the cis tautomer as a minor component. The zero-field splitting parameters and triplet sublevel populations indicate that the process is slow in the triplet state. The results obtained by EPR combined with photoselection and fluorescence anisotropy have been interpreted within a model which accounts for a fast trans-trans tautomerization promoted by a spin-vibronic coupling mechanism for intersystem crossing, with an even distribution of the two trans tautomers at liquid nitrogen temperatures for H2TPPS4-.

Correction: Solvent dependent triplet state delocalization in a co-facial porphyrin heterodimer.

Correction for 'Solvent dependent triplet state delocalization in a co-facial porphyrin heterodimer' by Susanna Ciuti et al., Phys. Chem. Chem. Phys., 2022, https://doi.org/10.1039/D2CP04291F.

Fullerene Complexation in a Hydrogen-Bonded Porphyrin Receptor via Induced-Fit: Cooperative Action of Tautomerization and C-H···π Interactions.

A supramolecular chiral hydrogen-bonded tetrameric aggregate possessing a large cavity and tetraarylporphyrin substituents was assembled using alternating 4H- and 2H-bonds between ureidopyrimidinone and isocytosine units, respectively. The aggregation mode was rationally shifted from social to narcissistic self-sorting by changing urea substituent size only. The H-bonded tetramer forms a strong complex with C60 guest, at the same time undergoing remarkable structural changes. Namely, the cavity adjusts to the guest via keto-to-enol tautomerization of the ureidopyrimidinone unit and as a result, porphyrin substituents move apart from each other in a scissor blade-like opening fashion. The rearrangement is accompanied by C-H···π interaction between the alkyl solubilizing groups and the nearby placed porphyrin π-systems. The latter interaction was found to be crucial for the guest complexation event, providing energetic compensation for otherwise costly tautomerization. We showed that only the systems possessing sufficiently long alkyl chains capable of interacting with a porphyrin ring are able to form a complex with C60. The structural rearrangement of the tetramer was quantitatively characterized by electron paramagnetic resonance pulsed dipolar spectroscopy measurements using photogenerated triplets of porphyrin and C60 as spin probes. Further exploring the C-H···π interaction as a decisive element for the C60 recognition, we investigated the guest-induced self-sorting phenomenon using scrambled tetramer assemblies composed of two types of monomers possessing alkyl chains of different lengths. The presence of the fullerene guest has enabled the selective scavenging of monomers capable of C-H···π interaction to form homo-tetrameric aggregates.

Solvent dependent triplet state delocalization in a co-facial porphyrin heterodimer.

The excited triplet state of a cofacial aluminum(III) porphyrin-phosphorus(V) porphyrin heterodimer is investigated using transient EPR spectroscopy and quantum chemical calculations. In the dimer, the two porphyrins are bound covalently to each other via a µ-oxo bond between the Al and P centres, which results in strong electronic interaction between the porphyrin rings. The spin polarized transient EPR spectrum of the dimer is narrower than the spectra of the constituent monomers and the magnitude of the zero-field splitting parameter D is solvent dependent, decreasing as the polarity of the solvent increases. The quantum chemical calculations show that the spin density of the triplet state is delocalized over both porphyrins, while magnetophotoselection measurements reveal that, in contrast to the value of D, the relative orientation of the ZFS axes and the excitation transition dipole moments are not solvent dependent. Together the results indicate that triplet state wavefunction is delocalized over both porphyrins and has a modest degree of charge-transfer character that increases with increasing solvent polarity. The sign of the spin polarization pattern of the dimer triplet state is opposite to that of the monomers. The positive sign of D predicted for the monomers and dimer by the quantum chemical calculations implies that the different signs of the spin polarization patterns is a result of a difference in the spin selectivity of the intersystem crossing.

Erythrosin B as a New Photoswitchable Spin Label for Light-Induced Pulsed EPR Dipolar Spectroscopy.

We present a new photoswitchable spin label for light-induced pulsed electron paramagnetic resonance dipolar spectroscopy (LiPDS), the photoexcited triplet state of erythrosin B (EB), which is ideal for biological applications. With this label, we perform an in-depth study of the orientational effects in dipolar traces acquired using the refocused laser-induced magnetic dipole technique to obtain information on the distance and relative orientation between the EB and nitroxide labels in a rigid model peptide, in good agreement with density functional theory predictions. Additionally, we show that these orientational effects can be averaged to enable an orientation-independent analysis to determine the distance distribution. Furthermore, we demonstrate the feasibility of these experiments above liquid nitrogen temperatures, removing the need for expensive liquid helium or cryogen-free cryostats. The variety of choices in photoswitchable spin labels and the affordability of the experiments are critical for LiPDS to become a widespread methodology in structural biology.

Magnetophotoselection in the Investigation of Excitonically Coupled Chromophores: The Case of the Water-Soluble Chlorophyll Protein.

A magnetophotoselection (MPS) investigation of the photoexcited triplet state of chlorophyll a both in a frozen organic solvent and in a protein environment, provided by the water-soluble chlorophyll protein (WSCP) of Lepidium virginicum, is reported. The MPS experiment combines the photoselection achieved by exciting with linearly polarized light with the magnetic selection of electron paramagnetic resonance (EPR) spectroscopy, allowing the determination of the relative orientation of the optical transition dipole moment and the zero-field splitting tensor axes in both environments. We demonstrate the robustness of the proposed methodology for a quantitative description of the excitonic interactions among pigments. The orientation of the optical transition dipole moments determined by the EPR analysis in WSCP, identified as an appropriate model system, are in excellent agreement with those calculated in the point-dipole approximation. In addition, MPS provides information on the electronic properties of the triplet state, localized on a single chlorophyll a pigment of the protein cluster, in terms of orientation of the zero-field splitting tensor axes in the molecular frame.

Light-induced pulsed dipolar EPR spectroscopy for distance and orientation analysis.

Measuring distances in biology at the molecular level is of great importance for understanding the structure and function of proteins, nucleic acids and other biological molecules and their complexes. Pulsed Dipolar Spectroscopy (PDS) offers advantages with respect to other methods as it is uniquely sensitive and specific to electronic spin centers and allows measurements in near-native conditions, comprising the in-cell environment. PDS methods measure the electron spin-spin dipolar interaction, therefore they require the presence of at least two paramagnetic centers, which are often stable radicals. Recent developments have introduced transient triplet states, photo-activated by a laser pulse, as spin labels and probes, thereby establishing a new family of techniques-Light-induced PDS (LiPDS). In this chapter, an overview of these methods is provided, looking at the chromophores that can be used for LiPDS and some of the technical aspects of the experiments. A guide to the choice of technique that can yield the best results, depending on the type of system studied and the information required, is provided. Examples of previous LiPDS studies of model systems and proteins are given. Characterization data for the chromophores used in these studies is tabulated to help selection of appropriate triplet state probes in future studies.

Primary donor triplet states of Photosystem I and II studied by Q-band pulse ENDOR spectroscopy.

The photoexcited triplet state of the "primary donors" in the two photosystems of oxygenic photosynthesis has been investigated by means of electron-nuclear double resonance (ENDOR) at Q-band (34 GHz). The data obtained represent the first set of 1H hyperfine coupling tensors of the 3P700 triplet state in PSI and expand the existing data set for 3P680. We achieved an extensive assignment of the observed electron-nuclear hyperfine coupling constants (hfcs) corresponding to the methine α-protons and the methyl group ß-protons of the chlorophyll (Chl) macrocycle. The data clearly confirm that in both photosystems the primary donor triplet is located on one specific monomeric Chl at cryogenic temperature. In comparison to previous transient ENDOR and pulse ENDOR experiments at standard X-band (9-10 GHz), the pulse Q-band ENDOR spectra demonstrate both improved signal-to-noise ratio and increased resolution. The observed ENDOR spectra for 3P700 and 3P680 differ in terms of the intensity loss of lines from specific methyl group protons, which is explained by hindered methyl group rotation produced by binding site effects. Contact analysis of the methyl groups in the PSI crystal structure in combination with the ENDOR analysis of 3P700 suggests that the triplet is located on the Chl a' (PA) in PSI. The results also provide additional evidence for the localization of 3P680 on the accessory ChlD1 in PSII.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

Orientation-Selective and Frequency-Correlated Light-Induced Pulsed Dipolar Spectroscopy.

We explore the potential of orientation-resolved pulsed dipolar spectroscopy (PDS) in light-induced versions of the experiment. The use of triplets as spin-active moieties for PDS offers an attractive tool for studying biochemical systems containing optically active cofactors. Cofactors are often rigidly bound within the protein structure, providing an accurate positional marker. The rigidity leads to orientation selection effects in PDS, which can be analyzed to give both distance and mutual orientation information. Herein we present a comprehensive analysis of the orientation selection of a full set of light-induced PDS experiments. We exploit the complementary information provided by the different light-induced techniques to yield atomic-level structural information. For the first time, we measure a 2D frequency-correlated laser-induced magnetic dipolar spectrum, and we are able to monitor the complete orientation dependence of the system in a single experiment. Alternatively, the summed spectrum enables an orientation-independent analysis to determine the distance distribution.

A distinctive pathway for triplet-triplet energy transfer photoprotection in fucoxanthin chlorophyll-binding proteins from Cyclotella meneghiniana.

Fucoxanthin chlorophyll-binding proteins (FCPs) are the major light-harvesting complexes of diatoms. In this work, FCPs isolated from Cyclotella meneghiniana have been studied by means of optically detected magnetic resonance (ODMR) and time-resolved electron paramagnetic resonance (TR-EPR), with the aim to characterize the photoprotective mechanism based on triplet-triplet energy transfer (TTET). The spectroscopic properties of the chromophores carrying the triplet state have been interpreted on the basis of a delved analysis of the recently solved crystallographic structures of FCP. The results point toward a photoprotective role for two fucoxanthin molecules exposed to the exterior of the FCP monomers. This shows that FCP has adopted a structural strategy different from that of related light-harvesting complexes from plants and other microalgae, in which the photoprotective role is carried out by two highly conserved carotenoids in the interior of the complex.

A charge transfer state induced by strong exciton coupling in a cofacial µ-oxo-bridged porphyrin heterodimer.

Photosensitizers with high energy, long lasting charge-transfer states are important components in systems designed for solar energy conversion by multistep electron transfer. Here, we show that in a push-pull type, µ-oxo-bridged porphyrin heterodimer composed of octaethylporphyrinatoaluminum(iii) and octaethylporphyrinatophosphorus(v), the strong excitonic coupling between the porphyrins and the different electron withdrawing abilities of Al(iii) and P(v) promote the formation of a high energy CT state. Using, an array of optical and magnetic resonance spectroscopic methods along with theoretical calculations, we demonstrate photodynamics of the heterodimer that involves the initial formation of a singlet CT which relaxes to a triplet CT state with a lifetime of ∼130 ps. The high-energy triplet CT state (3CT = 1.68 eV) lasts for nearly 105 µs prior to relaxing to the ground state.

Light-Induced Triplet-Triplet Electron Resonance Spectroscopy.

We present a new technique, light-induced triplet-triplet electron resonance spectroscopy (LITTER), which measures the dipolar interaction between two photoexcited triplet states, enabling both the distance and angular distributions between the two triplet moieties to be determined on a nanometer scale. This is demonstrated for a model bis-porphyrin peptide that renders dipolar traces with strong orientation selection effects. Using simulations and density functional theory calculations, we extract distance distributions and relative orientations of the porphyrin moieties, allowing the dominant conformation of the peptide in a frozen solution to be identified. LITTER removes the requirement of current light-induced electron spin resonance pulse dipolar spectroscopy techniques to have a permanent paramagnetic moiety, becoming more suitable for in-cell applications and facilitating access to distance determination in unmodified macromolecular systems containing photoexcitable moieties. LITTER also has the potential to enable direct comparison with Förster resonance energy transfer and combination with microscopy inside cells.

Electron spin polarization transfer induced by triplet-radical interactions in the weakly coupled regime.

We report the observation of electron spin polarization transfer from the triplet state of a porphyrin to a weakly coupled nitroxide radical in a mutant of human neuroglobin (NGB). The native iron-containing heme substrate of NGB has been substituted with Zn(ii) protoporphyrin IX and the nitroxide has been attached via site-directed spin labeling to the Cys120 residue. A reference synthetic polypeptide with free base tetraphenylporphyrin and a nitroxide bound to it is also studied. In both systems the nitroxide and the porphyrin are held at a fixed distance of approximately 2.4 nm. The transient EPR data of the NGB sample show that the triplet state of Zn(ii) protoporphyrin acquires significant net polarization, which is attributed to the dynamic Jahn-Teller effect. As the spin polarization of the protoporphyrin triplet state decays, a polarized EPR signal of the nitroxide arises. In contrast, the free base porphyrin in the reference polypeptide does not acquire net polarization and no polarization of the nitroxide label is observed. This is likely a result of the fact that the porphyrin is not Jahn-Teller active because of its lower symmetry. A perturbation theory treatment suggests that in the NGB sample, the polarization of the radical occurs by the transfer of net polarization from the triplet state. This process is also enhanced by the spectral broadening caused by the back and forth transitions associated with the dynamic Jahn-Teller effect. We propose that the novel transfer of polarization to the radical could be exploited to enhance the sensitivity of light-induced dipolar spectroscopy experiments.

Reversible triplet energy hopping in photo-excited molecules: A two-site model for the spin polarization.

The effect of reversible energy hopping between different local environments on the properties of spin-polarized excited states is investigated theoretically using a two-site model. The kinetic equations for the populations of the spin sublevels of the excited state are derived and then used to obtain analytical expressions for the evolution of the spin polarization of excited triplet states under specific conditions. The time dependence of the triplet state polarization patterns is also obtained by numerical solution of the kinetic equations. It is shown that the reversible energy hopping can lead to significant changes in the properties of the triplet state, including changes in the shape of the observed spectrum and, in some cases, the inversion of the sign of the polarization, the generation of the net polarization, and anisotropic spin-lattice relaxation. The relations between the parameters that can be observed experimentally by time-resolved electron paramagnetic resonance spectroscopy and the kinetic and dynamic parameters of the system are discussed.