ATCUN-like Copper Site in ßB2-Crystallin Plays a Protective Role in Cataract-Associated Aggregation.

Cataract is the leading cause of blindness worldwide, and it is caused by crystallin damage and aggregation. Senile cataractous lenses have relatively high levels of metals, while some metal ions can directly induce the aggregation of human γ-crystallins. Here, we evaluated the impact of divalent metal ions in the aggregation of human ßB2-crystallin, one of the most abundant crystallins in the lens. Turbidity assays showed that Pb2+, Hg2+, Cu2+, and Zn2+ ions induce the aggregation of ßB2-crystallin. Metal-induced aggregation is partially reverted by a chelating agent, indicating the formation of metal-bridged species. Our study focused on the mechanism of copper-induced aggregation of ßB2-crystallin, finding that it involves metal-bridging, disulfide-bridging, and loss of protein stability. Circular dichroism and electron paramagnetic resonance (EPR) revealed the presence of at least three Cu2+ binding sites in ßB2-crystallin, one of them with spectroscopic features typical for Cu2+ bound to an amino-terminal copper and nickel (ATCUN) binding motif, which is found in Cu transport proteins. The ATCUN-like Cu binding site is located at the unstructured N-terminus of ßB2-crystallin, and it could be modeled by a peptide with the first six residues in the protein sequence (NH2-ASDHQF-). Isothermal titration calorimetry indicates a nanomolar Cu2+ binding affinity for the ATCUN-like site. An N-truncated form of ßB2-crystallin is more susceptible to Cu-induced aggregation and is less thermally stable, indicating a protective role for the ATCUN-like site. EPR and X-ray absorption spectroscopy studies reveal the presence of a copper redox active site in ßB2-crystallin that is associated with metal-induced aggregation and formation of disulfide-bridged oligomers. Our study demonstrates metal-induced aggregation of ßB2-crystallin and the presence of putative copper binding sites in the protein. Whether the copper-transport ATCUN-like site in ßB2-crystallin plays a functional/protective role or constitutes a vestige from its evolution as a lens structural protein remains to be elucidated.

Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of ß-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

Molecular Dynamics and Structural Studies of Zinc Chloroquine Complexes.

Chloroquine (CQ) is a first-choice drug against malaria and autoimmune diseases. It has been co-administered with zinc against SARS-CoV-2 and soon dismissed because of safety issues. The structural features of Zn-CQ complexes and the effect of CQ on zinc distribution in cells are poorly known. In this study, state-of-the-art computations combined with experiments were leveraged to solve the structural determinants of zinc-CQ interactions in solution and the solid state. NMR, ESI-MS, and X-ray absorption and diffraction methods were combined with ab initio molecular dynamics calculations to address the kinetic lability of this complex. Within the physiological pH range, CQ binds Zn2+ through the quinoline ring nitrogen, forming [Zn(CQH)Clx(H2O)3-x](3+)-x (x = 0, 1, 2, and 3) tetrahedral complexes. The Zn(CQH)Cl3 species is stable at neutral pH and at high chloride concentrations typical of the extracellular medium, but metal coordination is lost at a moderately low pH as in the lysosomal lumen. The pentacoordinate complex [Zn(CQH)(H2O)4]3+ may exist in the absence of chloride. This in vitro/in silico approach can be extended to other metal-targeting drugs and bioinorganic systems.

Chloroquine disrupts zinc storage granules in primary Malpighian tubule cells of Drosophila melanogaster.

Contrasting reports exist in the literature regarding the effect of chloroquine treatment on cellular zinc uptake or secretion. Here, we tested the effect of chloroquine administration in the Drosophila model organism. We show that larvae grown on a diet supplemented with 2.5 mg/ml chloroquine lose up to 50% of their stored zinc and around 10% of their total potassium content. This defect in chloroquine-treated animals correlates with the appearance of abnormal autophagolysosomes in the principal cells of the Malpighian tubules, where zinc storage granules reside. We further show that the reported increase of Fluozin-3 fluorescence following treatment of cells with 300 µM chloroquine for 1 h may not reflect increased zinc accumulation, since a similar treatment in Madin-Darby canine kidney cells results in a 36% decrease in their total zinc content. Thus, chloroquine should not be considered a zinc ionophore. Zinc supplementation plus chloroquine treatment restored zinc content both in vivo and in vitro, without correcting autophagic or other ionic alterations, notably in potassium, associated with the chloroquine treatment. We suggest that chloroquine or hydroxychloroquine administration to patients could reduce intracellular zinc storage pools and be part of the drug's mechanism of action.

Tryptophan regulates Drosophila zinc stores.

Zinc deficiency is commonly attributed to inadequate absorption of the metal. Instead, we show that body zinc stores in Drosophila melanogaster depend on tryptophan consumption. Hence, a dietary amino acid regulates zinc status of the whole insect­a finding consistent with the widespread requirement of zinc as a protein cofactor. Specifically, the tryptophan metabolite kynurenine is released from insect fat bodies and induces the formation of zinc storage granules in Malpighian tubules, where 3-hydroxykynurenine and xanthurenic acid act as endogenous zinc chelators. Kynurenine functions as a peripheral zinc-regulating hormone and is converted into a 3-hydroxykynurenine­zinc­chloride complex, precipitating within the storage granules. Thus, zinc and the kynurenine pathway­well-known modulators of immunity, blood pressure, aging, and neurodegeneration­are physiologically connected.

The Type 1 Blue Copper Site: From Electron Transfer to Biological Function.

Cupredoxins host in their scaffold one of the most studied and interesting metal sites in biology: the type 1 (T1) or blue Cu center. Blue Cu proteins have evolved to play key roles in biological electron transfer and have the ability to react with a wide variety of redox partners. The inner coordination sphere of T1 Cu sites conserves two histidines and one cysteine with a short Cu-S(Cys) bond as ligands in a trigonal arrangement, with a variable axial ligand that modulates the electronic structure and reactivity. The structural, electronic and geometric features of T1 Cu centers provide the basis for a site that can be optimized by the protein structure for each biological function. This chapter highlights the properties that make this unique Cu center in biology an efficient and tunable electron transfer site. The contributions of the first coordination shell and the high covalency of the Cu-S(Cys) bond in the T1 Cu site to its distinctive geometric and spectroscopic features are discussed, as well as the role of the protein scaffold in imposing an 'entatic' state with a distorted tetrahedral geometry that minimizes geometric changes upon redox cycling. The analysis of naturally occurring perturbed blue Cu sites provides further insights into how the protein scaffold can tune the properties of the T1 Cu site. Blue Cu sites display a wide range of reduction potentials, as these are tuned to be consistent with their physiologically relevant electron donors and acceptors. The different properties of the protein matrix that play important roles in finetuning the reduction potential of T1 Cu sites are also discussed, including the nature of the axial ligand and outer coordination sphere effects. These concepts are further illustrated by the discussion of examples of biosynthetic blue Cu proteins. Finally, the different features of the T1 Cu site that make it an optimal site for electron transfer (ET) are discussed, in terms of Markus theory for intra- and inter-molecular ET. The active site in multicopper oxidases is used as an example to illustrate the contributions of the anisotropic covalency of the blue Cu site to an efficient ET, while the diverse reactivity of the T1 Cu sites in these enzymes is discussed to dissect the different properties provided by the protein that help tune these unique sites for biological ET.

Anion Binding and Oxidative Modification at the Molybdenum Cofactor of Formate Dehydrogenase from Rhodobacter capsulatus Studied by X-ray Absorption Spectroscopy.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N3-) or cyanate (OCN-). Azide provided best protection against O2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

Light-driven hydrogen evolution catalyzed by a cobaloxime catalyst incorporated in a MIL-101(Cr) metal-organic framework.

A cobaloxime H2 evolution catalyst with a hydroxo-functionalized pyridine ligand, Co(dmgH)2(4-HEP)Cl [dmgH = dimethylglyoxime, 4-HEP = 4-(2-hydroxyethyl)pyridine] was immobilized on a chromium terephthalate metal-organic framework (MOF), MIL-101(Cr), to construct a MOF-catalyst hybrid which displays good photocatalytic H2 evolution activity. The longevity of the cobaloxime catalyst is increased by MOF incorporation, but limited by the stability of the cobalt-pyridine bond under turnover conditions.

Kα X-ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S3 State.

The unique manganese-calcium catalyst in photosystem II (PSII) is the natural paragon for efficient light-driven water oxidation to yield O2. The oxygen-evolving complex (OEC) in the dark-stable state (S1) comprises a Mn4CaO4 core with five metal-bound water species. Binding and modification of the water molecules that are substrates of the water-oxidation reaction is mechanistically crucial but controversially debated. Two recent crystal structures of the OEC in its highest oxidation state (S3) show either a vacant Mn coordination site or a bound peroxide species. For purified PSII at room temperature, we collected Mn Kα X-ray emission spectra of the S0, S1, S2, and S3 intermediates in the OEC cycle, which were analyzed by comparison to synthetic Mn compounds, spectral simulations, and OEC models from density functional theory. Our results contrast both crystallographic structures. They indicate Mn oxidation in three S-transitions and suggest additional water binding at a previously open Mn coordination site. These findings exclude Mn reduction and render peroxide formation in S3 unlikely.

Inhibitory and Non-Inhibitory NH3 Binding at the Water-Oxidizing Manganese Complex of Photosystem II Suggests Possible Sites and a Rearrangement Mode of Substrate Water Molecules.

The identity and rearrangements of substrate water molecules in photosystem II (PSII) water oxidation are of great mechanistic interest and addressed herein by comprehensive analysis of NH4+/NH3 binding. Time-resolved detection of O2 formation and recombination fluorescence as well as Fourier transform infrared (FTIR) difference spectroscopy on plant PSII membrane particles reveals the following. (1) Partial inhibition in NH4Cl buffer occurs with a pH-independent binding constant of ∼25 mM, which does not result from decelerated O2 formation, but from complete blockage of a major PSII fraction (∼60%) after reaching the Mn(IV)4 (S3) state. (2) The non-inhibited PSII fraction advances through the reaction cycle, but modified nuclear rearrangements are suggested by FTIR difference spectroscopy. (3) Partial inhibition can be explained by anticooperative (mutually exclusive) NH3 binding to one inhibitory and one non-inhibitory site; these two sites may correspond to two water molecules terminally bound to the "dangling" Mn ion. (4) Unexpectedly strong modifications of the FTIR difference spectra suggest that in the non-inhibited PSII, ammonia binding obliterates the need for some of the nuclear rearrangements occurring in the S2-S3 transition as well as their reversal in the O2 formation transition, in line with the carousel mechanism [Askerka, M., et al. (2015) Biochemistry 54, 5783]. (5) We observe the same partial inhibition of PSII by NH4Cl also for thylakoid membranes prepared from mesophilic and thermophilic cyanobacteria, suggesting that the results described above are valid for plant and cyanobacterial PSII.

Electronic and molecular structure relations in diiron compounds mimicking the [FeFe]-hydrogenase active site studied by X-ray spectroscopy and quantum chemistry.

Synthetic diiron compounds of the general formula Fe2(µ-S2R)(CO)n(L)6-n (R = alkyl or aromatic groups; L = CN- or phosphines) are versatile models for the active-site cofactor of hydrogen turnover in [FeFe]-hydrogenases. A series of 18 diiron compounds, containing mostly a dithiolate bridge and terminal ligands of increasing complexity, was characterized by X-ray absorption and emission spectroscopy in combination with density functional theory. Fe K-edge absorption and Kß main-line emission spectra revealed the varying geometry and the low-spin state of the Fe(i) centers. Good agreement between experimental and calculated core-to-valence-excitation absorption and radiative valence-to-core-decay emission spectra revealed correlations between spectroscopic and structural features and provided access to the electronic configuration. Four main effects on the diiron core were identified, which were preferentially related to variation either of the dithiolate or of the terminal ligands. Alteration of the dithiolate bridge affected mainly the Fe-Fe bond strength, while more potent donor substitution and ligand field asymmetrization changed the metal charge and valence level localization. In contrast, cyanide ligation altered all relevant properties and, in particular, the frontier molecular orbital energies of the diiron core. Mutual benchmarking of experimental and theoretical parameters provides guidelines to verify the electronic properties of related diiron compounds.

Effective intermediate-spin iron in O2-transporting heme proteins.

Proteins carrying an iron-porphyrin (heme) cofactor are essential for biological O2 management. The nature of Fe-O2 bonding in hemoproteins is debated for decades. We used energy-sampling and rapid-scan X-ray Kß emission and K-edge absorption spectroscopy as well as quantum chemistry to determine molecular and electronic structures of unligated (deoxy), CO-inhibited (carboxy), and O2-bound (oxy) hemes in myoglobin (MB) and hemoglobin (HB) solutions and in porphyrin compounds at 20-260 K. Similar metrical and spectral features revealed analogous heme sites in MB and HB and the absence of low-spin (LS) to high-spin (HS) conversion. Amplitudes of Kß main-line emission spectra were directly related to the formal unpaired Fe(d) spin count, indicating HS Fe(II) in deoxy and LS Fe(II) in carboxy. For oxy, two unpaired Fe(d) spins and, thus by definition, an intermediate-spin iron center, were revealed by our static and kinetic X-ray data, as supported by (time-dependent) density functional theory and complete-active-space self-consistent-field calculations. The emerging Fe-O2 bonding situation includes in essence a ferrous iron center, minor superoxide character of the noninnocent ligand, significant double-bond properties of the interaction, and three-center electron delocalization as in ozone. It resolves the apparently contradictory classical models of Pauling, Weiss, and McClure/Goddard into a unifying view of O2 bonding, tuned toward reversible oxygen transport.

Spontaneous Si-C bond cleavage in (TriphosSi)-nickel complexes.

Herein, we report on the versatile reactions of CH3C(CH2PPh2)3 as well as CH3Si(CH2PPh2)3 derived Ni-complexes. While Ni[CH3C(CH2PPh2)3] complexes reveal high stability, the Ni[CH3Si(CH2PPh2)3] analogs show rapid decomposition at room temperature and afford the unprecedented pseudo-tetrahedral phosphino methanide complex 5. We provide a detailed electronic structure of 5 from X-ray absorption and emission spectroscopy data analysis in combination with DFT calculations, as well as from comparison with structurally related complexes. A mechanistic study for the formation of complex 5 by reaction with BF4- is presented, based on a comparison of experimental data with quantum chemical calculations. We also show a simple route towards isolable Ni(i)-complexes on the gram scale.

Protonation State of MnFe and FeFe Cofactors in a Ligand-Binding Oxidase Revealed by X-ray Absorption, Emission, and Vibrational Spectroscopy and QM/MM Calculations.

Enzymes with a dimetal-carboxylate cofactor catalyze reactions among the top challenges in chemistry such as methane and dioxygen (O2) activation. Recently described proteins bind a manganese-iron cofactor (MnFe) instead of the classical diiron cofactor (FeFe). Determination of atomic-level differences of homo- versus hetero-bimetallic cofactors is crucial to understand their diverse redox reactions. We studied a ligand-binding oxidase from the bacterium Geobacillus kaustophilus (R2lox) loaded with a FeFe or MnFe cofactor, which catalyzes O2 reduction and an unusual tyrosine-valine ether cross-link formation, as revealed by X-ray crystallography. Advanced X-ray absorption, emission, and vibrational spectroscopy methods and quantum chemical and molecular mechanics calculations provided relative Mn/Fe contents, X-ray photoreduction kinetics, metal-ligand bond lengths, metal-metal distances, metal oxidation states, spin configurations, valence-level degeneracy, molecular orbital composition, nuclear quadrupole splitting energies, and vibrational normal modes for both cofactors. A protonation state with an axial water (H2O) ligand at Mn or Fe in binding site 1 and a metal-bridging hydroxo group (µOH) in a hydrogen-bonded network is assigned. Our comprehensive picture of the molecular, electronic, and dynamic properties of the cofactors highlights reorientation of the unique axis along the Mn-OH2 bond for the Mn1(III) Jahn-Teller ion but along the Fe-µOH bond for the octahedral Fe1(III). This likely corresponds to a more positive redox potential of the Mn(III)Fe(III) cofactor and higher proton affinity of its µOH group. Refined model structures for the Mn(III)Fe(III) and Fe(III)Fe(III) cofactors are presented. Implications of our findings for the site-specific metalation of R2lox and performance of the O2 reduction and cross-link formation reactions are discussed.