Understanding the function of Mn ions in biological and chemical redox catalysis requires precise knowledge of their electronic structure. X-ray emission spectroscopy (XES) is an emerging technique with a growing application to biological and biomimetic systems. Here, we report an improved, cost-effective spectrometer used to analyze two biomimetic coordination compounds, [MnIV(OH)2(Me2EBC)]2+ and [MnIV(O)(OH)(Me2EBC)]+, the second of which contains a key MnIV═O structural fragment. Despite having the same formal oxidation state (MnIV) and tetradentate ligands, XES spectra from these two compounds demonstrate different electronic structures. Experimental measurements and DFT calculations yield different localized spin densities for the two complexes resulting from MnIV–OH conversion to MnIV═O. The relevance of the observed spectroscopic changes is discussed for applications in analyzing complex biological systems such as photosystem II. A model of the S3 intermediate state of photosystem II containing a MnIV═O fragment is compared to recent time-resolved X-ray diffraction data of the same state.
Abstract Fulfilling a bevy of biological roles, copper is an essential metal for healthy brain function. Cu dyshomeostasis has been demonstrated to be involved in some neurological conditions including Menkes and Alzheimer's diseases. We have previously reported localized Cu-rich aggregates in astrocytes of the subventricular zone (SVZ) in rodent brains with Cu concentrations in the hundreds of millimolar. Metallothionein, a cysteine-rich protein critical to metal homeostasis and known to participate in a variety of neuroprotective and neuroregenerative processes, was proposed as a binding protein. Here, we present an analysis of metallothionein(1,2) knockout (MTKO) mice and age-matched controls using X-ray fluorescence microscopy. In large structures such as the corpus callosum, cortex, and striatum, there is no significant difference in Cu, Fe, or Zn concentrations in \MTKO\ mice compared to age-matched controls. In the astrocyte-rich subventricular zone where Cu-rich aggregates reside, approximately 1/3 as many Cu-rich aggregates persist in \MTKO\ mice resulting in a decrease in periventricular Cu concentration. Aggregates in both wild-type and \MTKO\ mice show \XANES\ spectra characteristic of CuxSy multimetallic clusters and have similar [S]/[Cu] ratios. Consistent with assignment as a CuxSy multimetallic cluster, the astrocyte-rich \SVZ\ of both \MTKO\ and wild-type mice exhibit autofluorescent bodies, though \MTKO\ mice exhibit fewer. Furthermore, \XRF\ imaging of Au-labeled lysosomes and ubiquitin demonstrates a lack of co-localization with Cu-rich aggregates suggesting they are not involved in a degradation pathway. Overall, these data suggest that Cu in aggregates is bound by either metallothionein-3 or a yet unknown protein similar to metallothionein.
Galectins are proteins involved in diverse cellular contexts due to their capacity to decipher and respond to the information encoded by beta-galactoside sugars. In particular, human galectin-4, normally expressed in the healthy gastrointestinal tract, displays differential expression in cancerous tissues and is considered a potential drug target for liver and lung cancer. Galectin-4 is a tandem-repeat galectin characterized by two carbohydrate recognition domains connected by a linker-peptide. Despite their relevance to cell function and pathogenesis, structural characterization of full-length tandem-repeat galectins has remained elusive. Here, we investigate galectin-4 using X-ray crystallography, small-and wide-angle X-ray scattering, molecular modelling, molecular dynamics simulations, and differential scanning fluorimetry assays and describe for the first time a structural model for human galectin-4. Our results provide insight into the structural role of the linker-peptide and shed light on the dynamic characteristics of the mechanism of carbohydrate recognition among tandem-repeat galectins.
The functional structure of the Oxygen Evolving Complex, the Mn4Ca cluster of Photosystem II, a critical component of natural photosynthesis, was analyzed at room temperature by EXAFS. An experimental improvement in the form of a spinning sample holder allowed us to efficiently counteract the severe X-ray damage sensitivity of Photosystem II to obtain high quality data subsequently used for a systematic evaluation of atomistic S1 state models. We investigated the accuracy of models collected by both conventional and femtosecond XRD at 1.9 and 1.95 Å resolution, respectively, as well as DFT-based models, to determine the structure most representative of experimental data. Additionally, room temperature EXAFS results do not show a visible reduction in the intensity of the k-space oscillations, supporting a rigid structure of the Mn4Ca cluster at room temperature.
Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.
It is well-known that biological samples undergo X-ray-induced degradation. One of the fastest occurring X-ray-induced processes involves redox modifications (reduction or oxidation) of redox-active cofactors in proteins. Here we analyze room-temperature data on the photoreduction of Mn ions in the oxygen-evolving complex (OEC) of photosystem II, one of the most radiation damage-sensitive proteins and a key constituent of natural photosynthesis in plants, green algae, and cyanobacteria. Time-resolved X-ray emission spectroscopy with wavelength-dispersive detection was used to collect data on the progression of X-ray-induced damage. A kinetic model was developed to fit experimental results, and the rate constant for the reduction of OEC Mn(III) and Mn(IV) ions by solvated electrons was determined. From this model, the possible kinetics of X-ray-induced damage at a variety of experimental conditions, such as different rates of dose deposition as well as different excitation wavelengths, can be inferred. We observed a trend of increasing dosage threshold prior to the onset of X-ray-induced damage with increasing rates of dose deposition. This trend suggests that experimentation with higher rates of dose deposition is beneficial for measurements of biological samples sensitive to radiation damage, particularly at pink beam and X-ray free electron laser sources.
Redox-inactive metal ions play pivotal roles in regulating the reactivities of high-valent metal-oxo species in a variety of enzymatic and chemical reactions. A mononuclear non-heme Mn(IV)-oxo complex bearing a pentadentate N5 ligand has been synthesized and used in the synthesis of a Mn(IV)-oxo complex binding scandium ions. The Mn(IV)-oxo complexes were characterized with various spectroscopic methods. The reactivities of the Mn(IV)-oxo complex are markedly influenced by binding of Sc(3+) ions in oxidation reactions, such as a ~2200-fold increase in the rate of oxidation of thioanisole (i.e., oxygen atom transfer) but a ~180-fold decrease in the rate of C-H bond activation of 1,4-cyclohexadiene (i.e., hydrogen atom transfer). The present results provide the first example of a non-heme Mn(IV)-oxo complex binding redox-inactive metal ions that shows a contrasting effect of the redox-inactive metal ions on the reactivities of metal-oxo species in the oxygen atom transfer and hydrogen atom transfer reactions.
The paradigm of "detection-before-destruction" was tested for a metalloprotein complex exposed at room temperature to the high x-ray flux typical of third generation synchrotron sources. Following the progression of the x-ray induced damage by Mn Kβ x-ray emission spectroscopy, we demonstrated the feasibility of collecting room temperature data on the electronic structure of native Photosystem II, a trans-membrane metalloprotein complex containing a Mn(4)Ca cluster. The determined non-damaging observation timeframe (about 100 milliseconds using continuous monochromatic beam, deposited dose 1*10(7) photons/µm(2) or 1.3*10(4) Gy, and 66 microseconds in pulsed mode using pink beam, deposited dose 4*10(7) photons/µm(2) or 4.2*10(4) Gy) is sufficient for the analysis of this protein's electron dynamics and catalytic mechanism at room temperature. Reported time frames are expected to be representative for other metalloproteins. The described instrumentation, based on the short working distance dispersive spectrometer, and experimental methodology is broadly applicable to time-resolved x-ray emission analysis at synchrotron and x-ray free-electron laser light sources.
A mononuclear non-heme manganese(IV)-oxo complex has been synthesized and characterized using various spectroscopic methods. The Mn(IV)-oxo complex shows high reactivity in oxidation reactions, such as C-H bond activation, oxidations of olefins, alcohols, sulfides, and aromatic compounds, and N-dealkylation. In C-H bond activation, the Mn(IV)-oxo complex can activate C-H bonds as strong as those in cyclohexane. It is proposed that C-H bond activation by the non-heme Mn(IV)-oxo complex does not occur via an oxygen-rebound mechanism. The electrophilic character of the non-heme Mn(IV)-oxo complex is demonstrated by a large negative ρ value of -4.4 in the oxidation of para-substituted thioanisoles.