Drosophila Evi5 is a critical regulator of intracellular iron transport via transferrin and ferritin interactions.

Vesicular transport is essential for delivering cargo to intracellular destinations. Evi5 is a Rab11-GTPase-activating protein involved in endosome recycling. In humans, Evi5 is a high-risk locus for multiple sclerosis, a debilitating disease that also presents with excess iron in the CNS. In insects, the prothoracic gland (PG) requires entry of extracellular iron to synthesize steroidogenic enzyme cofactors. The mechanism of peripheral iron uptake in insect cells remains controversial. We show that Evi5-depletion in the Drosophila PG affected vesicle morphology and density, blocked endosome recycling and impaired trafficking of transferrin-1, thus disrupting heme synthesis due to reduced cellular iron concentrations. We show that ferritin delivers iron to the PG as well, and interacts physically with Evi5. Further, ferritin-injection rescued developmental delays associated with Evi5-depletion. To summarize, our findings show that Evi5 is critical for intracellular iron trafficking via transferrin-1 and ferritin, and implicate altered iron homeostasis in the etiology of multiple sclerosis.

Iron Kß X-ray Emission Spectroscopy: The Origin of Spectral Features from Atomic to Molecular Systems Using Multi-configurational Calculations.

Kß X-ray emission spectroscopy (XES) is widely used to fingerprint the local spin of transition-metal ions, including in pump-probe experiments, to identify excited states or in chemical and biological reactions to characterize short-lived intermediates. In this study, the spectra of ferrous and ferric complexes for various spin states were measured experimentally and described theoretically through restricted active space (RAS) calculations including dynamic correlations. Through the RAS calculations from simple atomic models to complex molecular systems, spectral effects such as the exchange interactions, crystal-field strength, and covalent orbital mixing were evaluated and discussed. The calculations find that only the spectral features of low-spin cases show a dependence on the crystal-field strength, particularly for ferrous low spin. The effect of the covalent orbital mixing strength on the first moment of the Kß1,3 main line and the Kß1,3-Kß' energy splitting is quantitatively described. Clear relationships are found within a given nominal spin but less between different spin states, which calls for careful selection of reference spectra in future experiments. This study further advances our understanding of the correlation between changes in experimental spectral features and their corresponding electronic structure information.

Solution phase high repetition rate laser pump x-ray probe picosecond hard x-ray spectroscopy at the Stanford Synchrotron Radiation Lightsource.

We present a dedicated end-station for solution phase high repetition rate (MHz) picosecond hard x-ray spectroscopy at beamline 15-2 of the Stanford Synchrotron Radiation Lightsource. A high-power ultrafast ytterbium-doped fiber laser is used to photoexcite the samples at a repetition rate of 640 kHz, while the data acquisition operates at the 1.28 MHz repetition rate of the storage ring recording data in an alternating on-off mode. The time-resolved x-ray measurements are enabled via gating the x-ray detectors with the 20 mA/70 ps camshaft bunch of SPEAR3, a mode available during the routine operations of the Stanford Synchrotron Radiation Lightsource. As a benchmark study, aiming to demonstrate the advantageous capabilities of this end-station, we have conducted picosecond Fe K-edge x-ray absorption spectroscopy on aqueous [FeII(phen)3]2+, a prototypical spin crossover complex that undergoes light-induced excited spin state trapping forming an electronic excited state with a 0.6-0.7 ns lifetime. In addition, we report transient Fe Kß main line and valence-to-core x-ray emission spectra, showing a unique detection sensitivity and an excellent agreement with model spectra and density functional theory calculations, respectively. Notably, the achieved signal-to-noise ratio, the overall performance, and the routine availability of the developed end-station have enabled a systematic time-resolved science program using the monochromatic beam at the Stanford Synchrotron Radiation Lightsource.

AquaScouts: ePROs Implemented as a Serious Game for Children With Cancer to Support Palliative Care.

MyPal is a European initiative focusing on the use of the electronic patient reported outcome (ePRO) measures to enhance patient engagement in palliative cancer care via digital self-reporting palliative care for patients with cancer. As a part of its approach, MyPal also focuses on pediatric patients, implementing a specific digital health platform including a serious game to facilitate the reporting of the symptoms and overall status regarding their quality of life (QoL). To this end, the reduction of psychological burden related to frequent reporting, a.k.a. as "reporting fatigue" has been identified as a priority. In this study, we present the MyPal-CHILD platform, emphasizing on the serious game named AquaScouts and its key design decisions, while also emphasizing on the respective challenges. More specifically, we provide insights on the participatory design approach applied during the design of the platform and the high-level goals defined based on end-user input. In addition, the validation process applied before the use of the platform under real-world conditions is also presented. Finally, we discuss a number of challenges and the prospects of deploying eHealth interventions to support palliative care.

Effect of 3d/4p Mixing on 1s2p Resonant Inelastic X-ray Scattering: Electronic Structure of Oxo-Bridged Iron Dimers.

1s2p resonant inelastic X-ray scattering (1s2p RIXS) has proven successful in the determination of the differential orbital covalency (DOC, the amount of metal vs ligand character in each d molecular orbital) of highly covalent centrosymmetric iron environments including heme models and enzymes. However, many reactive intermediates have noncentrosymmetric environments, e.g., the presence of strong metal-oxo bonds, which results in the mixing of metal 4p character into the 3d orbitals. This leads to significant intensity enhancement in the metal K-pre-edge and as shown here, the associated 1s2p RIXS features, which impact their insight into electronic structure. Binuclear oxo bridged high spin Fe(III) complexes are used to determine the effects of 4p mixing on 1s2p RIXS spectra. In addition to developing the analysis of 4p mixing on K-edge XAS and 1s2p RIXS data, this study explains the selective nature of the 4p mixing that also enhances the analysis of L-edge XAS intensity in terms of DOC. These 1s2p RIXS biferric model studies enable new structural insight from related data on peroxo bridged biferric enzyme intermediates. The dimeric nature of the oxo bridged Fe(III) complexes further results in ligand-to-ligand interactions between the Fe(III) sites and angle dependent features just above the pre-edge that reflect the superexchange pathway of the oxo bridge. Finally, we present a methodology that enables DOC to be obtained when L-edge XAS is inaccessible and only 1s2p RIXS experiments can be performed as in many metalloenzyme intermediates in solution.

Short-lived metal-centered excited state initiates iron-methionine photodissociation in ferrous cytochrome c.

The dynamics of photodissociation and recombination in heme proteins represent an archetypical photochemical reaction widely used to understand the interplay between chemical dynamics and reaction environment. We report a study of the photodissociation mechanism for the Fe(II)-S bond between the heme iron and methionine sulfur of ferrous cytochrome c. This bond dissociation is an essential step in the conversion of cytochrome c from an electron transfer protein to a peroxidase enzyme. We use ultrafast X-ray solution scattering to follow the dynamics of Fe(II)-S bond dissociation and 1s3p (Kß) X-ray emission spectroscopy to follow the dynamics of the iron charge and spin multiplicity during bond dissociation. From these measurements, we conclude that the formation of a triplet metal-centered excited state with anti-bonding Fe(II)-S interactions triggers the bond dissociation and precedes the formation of the metastable Fe high-spin quintet state.

Soft X-ray spectroscopy with transition-edge sensors at Stanford Synchrotron Radiation Lightsource beamline 10-1.

We present results obtained with a new soft X-ray spectrometer based on transition-edge sensors (TESs) composed of Mo/Cu bilayers coupled to bismuth absorbers. This spectrometer simultaneously provides excellent energy resolution, high detection efficiency, and broadband spectral coverage. The new spectrometer is optimized for incident X-ray energies below 2 keV. Each pixel serves as both a highly sensitive calorimeter and an X-ray absorber with near unity quantum efficiency. We have commissioned this 240-pixel TES spectrometer at the Stanford Synchrotron Radiation Lightsource beamline 10-1 (BL 10-1) and used it to probe the local electronic structure of sample materials with unprecedented sensitivity in the soft X-ray regime. As mounted, the TES spectrometer has a maximum detection solid angle of 2 × 10-3 sr. The energy resolution of all pixels combined is 1.5 eV full width at half maximum at 500 eV. We describe the performance of the TES spectrometer in terms of its energy resolution and count-rate capability and demonstrate its utility as a high throughput detector for synchrotron-based X-ray spectroscopy. Results from initial X-ray emission spectroscopy and resonant inelastic X-ray scattering experiments obtained with the spectrometer are presented.

Manipulating charge transfer excited state relaxation and spin crossover in iron coordination complexes with ligand substitution.

Developing light-harvesting and photocatalytic molecules made with iron could provide a cost effective, scalable, and environmentally benign path for solar energy conversion. To date these developments have been limited by the sub-picosecond metal-to-ligand charge transfer (MLCT) electronic excited state lifetime of iron based complexes due to spin crossover - the extremely fast intersystem crossing and internal conversion to high spin metal-centered excited states. We revitalize a 30 year old synthetic strategy for extending the MLCT excited state lifetimes of iron complexes by making mixed ligand iron complexes with four cyanide (CN-) ligands and one 2,2'-bipyridine (bpy) ligand. This enables MLCT excited state and metal-centered excited state energies to be manipulated with partial independence and provides a path to suppressing spin crossover. We have combined X-ray Free-Electron Laser (XFEL) Kß hard X-ray fluorescence spectroscopy with femtosecond time-resolved UV-visible absorption spectroscopy to characterize the electronic excited state dynamics initiated by MLCT excitation of [Fe(CN)4(bpy)]2-. The two experimental techniques are highly complementary; the time-resolved UV-visible measurement probes allowed electronic transitions between valence states making it sensitive to ligand-centered electronic states such as MLCT states, whereas the Kß fluorescence spectroscopy provides a sensitive measure of changes in the Fe spin state characteristic of metal-centered excited states. We conclude that the MLCT excited state of [Fe(CN)4(bpy)]2- decays with roughly a 20 ps lifetime without undergoing spin crossover, exceeding the MLCT excited state lifetime of [Fe(2,2'-bipyridine)3]2+ by more than two orders of magnitude.

Tracking excited-state charge and spin dynamics in iron coordination complexes.

Crucial to many light-driven processes in transition metal complexes is the absorption and dissipation of energy by 3d electrons. But a detailed understanding of such non-equilibrium excited-state dynamics and their interplay with structural changes is challenging: a multitude of excited states and possible transitions result in phenomena too complex to unravel when faced with the indirect sensitivity of optical spectroscopy to spin dynamics and the flux limitations of ultrafast X-ray sources. Such a situation exists for archetypal polypyridyl iron complexes, such as [Fe(2,2'-bipyridine)3](2+), where the excited-state charge and spin dynamics involved in the transition from a low- to a high-spin state (spin crossover) have long been a source of interest and controversy. Here we demonstrate that femtosecond resolution X-ray fluorescence spectroscopy, with its sensitivity to spin state, can elucidate the spin crossover dynamics of [Fe(2,2'-bipyridine)3](2+) on photoinduced metal-to-ligand charge transfer excitation. We are able to track the charge and spin dynamics, and establish the critical role of intermediate spin states in the crossover mechanism. We anticipate that these capabilities will make our method a valuable tool for mapping in unprecedented detail the fundamental electronic excited-state dynamics that underpin many useful light-triggered molecular phenomena involving 3d transition metal complexes.

Metal-ligand covalency of iron complexes from high-resolution resonant inelastic X-ray scattering.

Data from Kα resonant inelastic X-ray scattering (RIXS) have been used to extract electronic structure information, i.e., the covalency of metal-ligand bonds, for four iron complexes using an experimentally based theoretical model. Kα RIXS involves resonant 1s→3d excitation and detection of the 2p→1s (Kα) emission. This two-photon process reaches similar final states as single-photon L-edge (2p→3d) X-ray absorption spectroscopy (XAS), but involves only hard X-rays and can therefore be used to get high-resolution L-edge-like spectra for metal proteins, solution catalysts and their intermediates. To analyze the information content of Kα RIXS spectra, data have been collected for four characteristic σ-donor and π-back-donation complexes: ferrous tacn [Fe(II)(tacn)2]Br2, ferrocyanide [Fe(II)(CN)6]K4, ferric tacn [Fe(III)(tacn)2]Br3 and ferricyanide [Fe(III)(CN)6]K3. From these spectra metal-ligand covalencies can be extracted using a charge-transfer multiplet model, without previous information from the L-edge XAS experiment. A direct comparison of L-edge XAS and Kα RIXS spectra show that the latter reaches additional final states, e.g., when exciting into the e(g) (σ*) orbitals, and the splitting between final states of different symmetry provides an extra dimension that makes Kα RIXS a more sensitive probe of σ-bonding. Another key difference between L-edge XAS and Kα RIXS is the π-back-bonding features in ferro- and ferricyanide that are significantly more intense in L-edge XAS compared to Kα RIXS. This shows that two methods are complementary in assigning electronic structure. The Kα RIXS approach can thus be used as a stand-alone method, in combination with L-edge XAS for strongly covalent systems that are difficult to probe by UV/vis spectroscopy, or as an extension to conventional absorption spectroscopy for a wide range of transition metal enzymes and catalysts.

Iron L-edge X-ray absorption spectroscopy of oxy-picket fence porphyrin: experimental insight into Fe-O2 bonding.

The electronic structure of the Fe-O(2) center in oxy-hemoglobin and oxy-myoglobin is a long-standing issue in the field of bioinorganic chemistry. Spectroscopic studies have been complicated by the highly delocalized nature of the porphyrin, and calculations require interpretation of multideterminant wave functions for a highly covalent metal site. Here, iron L-edge X-ray absorption spectroscopy, interpreted using a valence bond configuration interaction multiplet model, is applied to directly probe the electronic structure of the iron in the biomimetic Fe-O(2) heme complex [Fe(pfp)(1-MeIm)O(2)] (pfp ("picket fence porphyrin") = meso-tetra(α,α,α,α-o-pivalamidophenyl)porphyrin or TpivPP). This method allows separate estimates of σ-donor, π-donor, and π-acceptor interactions through ligand-to-metal charge transfer and metal-to-ligand charge transfer mixing pathways. The L-edge spectrum of [Fe(pfp)(1-MeIm)O(2)] is further compared to those of [Fe(II)(pfp)(1-MeIm)(2)], [Fe(II)(pfp)], and [Fe(III)(tpp)(ImH)(2)]Cl (tpp = meso-tetraphenylporphyrin) which have Fe(II)S = 0, Fe(II)S = 1, and Fe(III)S = 1/2 ground states, respectively. These serve as references for the three possible contributions to the ground state of oxy-pfp. The Fe-O(2) pfp site is experimentally determined to have both significant σ-donation and a strong π-interaction of the O(2) with the iron, with the latter having implications with respect to the spin polarization of the ground state.