Seven Steps of Alternating Electron and Proton Transfer in Photosystem II Water Oxidation Traced by Time-Resolved Photothermal Beam Deflection at Improved Sensitivity

The intricate orchestration of electron transfer (ET) and proton transfer (PT) at the Mn₄CaO_<i>n</i>-cluster of photosystem II (PSII) is mechanistically pivotal but clearly insufficiently understood. Preparations of PSII membrane particles were investigated using a kinetically competent and sensitive method, photothermal beam deflection (PBD), to monitor apparent volume changes of the PSII protein. Driven by nanosecond laser flashes, the PSII was synchronously stepped through its water-oxidation cycle involving four (semi)­stable states (S_0, S₁, S₂, and S₃) and minimally three additional transiently formed intermediates. The PBD approach was optimized as compared to our previous experiments, resulting in superior signal quality and resolution of more reaction steps. Now seven transitions were detected and attributed, according to the H/D-exchange, temperature, and pH effects on their time constants, to ET or PT events. The ET steps oxidizing the Mn₄CaO_<i>n</i> cluster in the S₂ → S₃ and S₀ → S₁ transitions, a biphasic PT prior to the O₂-evolving reaction, as well as the reoxidation of the primary quinone acceptor (Q_A^–) at the PSII acceptor side were detected for the first time by PBD. The associated volume changes involve (i) initial formation of charged groups resulting in contraction assignable to electrostriction, (ii) volume contraction explainable by reduced metal–ligand distances upon manganese oxidation, and (iii) charge-compensating proton removal resulting in volume expansion due to electrostriction reversal. These results support a reaction cycle of water oxidation exhibiting alternate ET and PT steps. An extended kinetic scheme for the O₂-evolving S₃ ⇒ S₀ transition is proposed, which includes crucial structural and protonic events

Abrupt versus Gradual Spin-Crossover in Fe^II(phen)₂(NCS)₂ and Fe^III(dedtc)₃ Compared by X‑ray Absorption and Emission Spectroscopy and Quantum-Chemical Calculations

Molecular spin-crossover (SCO) compounds are attractive for information storage and photovoltaic technologies. We compared two prototypic SCO compounds with Fe^IIN₆ (<b>1</b>, [Fe­(phen)₂(NCS)₂], with phen = 1,10-phenanthroline) or Fe^IIIS₆ (<b>2</b>, [Fe­(dedtc)₃], with dedtc = <i>N</i>,<i>N</i>′-diethyldithiocarbamate) centers, which show abrupt (<b>1</b>) or gradual (<b>2</b>) thermally induced SCO, using K-edge X-ray absorption and Kβ emission spectroscopy (XAS/XES) in a 8–315 K temperature range, single-crystal X-ray diffraction (XRD), and density functional theory (DFT). Core-to-valence and valence-to-core electronic transitions in the XAS/XES spectra and bond lengths change from XRD provided benchmark data, verifying the adequacy of the TPSSh/TZVP DFT approach for the description of low-spin (LS) and high-spin (HS) species. Determination of the spin densities, charge distributions, bonding descriptors, and valence-level configurations, as well as similar experimental and calculated enthalpy changes (Δ<i>H</i>), suggested that the varying metal–ligand bonding properties and deviating electronic structures converge to similar enthalpic contributions to the free-energy change (Δ<i>G</i>) and thus presumably are not decisive for the differing SCO behavior of <b>1</b> and <b>2</b>. Rather, SCO seems to be governed by vibrational contributions to the entropy changes (Δ<i>S</i>) in both complexes. Intra- and intermolecular interactions in crystals of <b>1</b> and <b>2</b> were identified by atoms-in-molecules analysis. Thermal excitation of individual dedtc ligand vibrations accompanies the gradual SCO in <b>2</b>. In contrast, extensive inter- and intramolecular phen/NCS vibrational mode coupling may be an important factor in the cooperative SCO behavior of <b>1</b>

Electronic Structure of an [FeFe] Hydrogenase Model Complex in Solution Revealed by X‑ray Absorption Spectroscopy Using Narrow-Band Emission Detection

High-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection, supported by density functional theory calculations (XAES-DFT), was used to study a model complex, ([Fe₂(μ-adt)­(CO)₄(PMe₃)₂] (<b>1</b>, adt = S–CH₂–(NCH₂Ph)–CH₂–S), of the [FeFe] hydrogenase active site. For <b>1</b> in powder material (<b>1</b>_powder), in MeCN solution (<b>1</b>′), and in its three protonated states (<b>1H</b>, <b>1Hy</b>, <b>1HHy</b>; <b>H</b> denotes protonation at the adt–N and <b>Hy</b> protonation of the Fe–Fe bond to form a bridging metal hydride), relations between the molecular structures and the electronic configurations were determined. EXAFS analysis and DFT geometry optimization suggested prevailing rotational isomers in MeCN, which were similar to the crystal structure or exhibited rotation of the (CO) ligands at Fe1 (<b>1</b>_CO, <b>1Hy</b>_CO) and in addition of the phenyl ring (<b>1H</b>_CO,Ph, <b>1HHy</b>_CO,Ph), leading to an elongated solvent-exposed Fe–Fe bond. Isomer formation, adt–N protonation, and hydride binding caused spectral changes of core-to-valence (pre-edge of the Fe K-shell absorption) and of valence-to-core (Kß^2,5 emission) electronic transitions, and of Kα RIXS data, which were quantitatively reproduced by DFT. The study reveals (1) the composition of molecular orbitals, for example, with dominant Fe-d character, showing variations in symmetry and apparent oxidation state at the two Fe ions and a drop in MO energies by ∼1 eV upon each protonation step, (2) the HOMO–LUMO energy gaps, of ∼2.3 eV for <b>1</b>_powder and ∼2.0 eV for <b>1</b>′, and (3) the splitting between iron d­(<i>z</i>²) and d­(<i>x</i>²–<i>y</i>²) levels of ∼0.5 eV for the nonhydride and ∼0.9 eV for the hydride states. Good correlations of reduction potentials to LUMO energies and oxidation potentials to HOMO energies were obtained. Two routes of facilitated bridging hydride binding thereby are suggested, involving ligand rotation at Fe1 for <b>1Hy</b>_CO or adt–N protonation for <b>1HHy</b>_CO,Ph. XAES-DFT thus enables verification of the effects of ligand substitutions in solution for guided improvement of [FeFe] catalysts

Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X‑ray Absorption Spectroscopy

Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium <i>Rhodobacter capsulatus</i>, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5–10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo­(VI) ion with each one MPT, MoO, Mo–O^–, and MoS ligand, and a weak Mo–O­(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (p<i>K</i> ∼ 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (p<i>K</i> ∼ 8.8) is an inhibitor at alkaline pH. A similar acidic p<i>K</i> for the WT and Q179A variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo–OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation

Site-Selective X-ray Spectroscopy on an Asymmetric Model Complex of the [FeFe] Hydrogenase Active Site

The active site for hydrogen production in [FeFe] hydrogenase comprises a diiron unit. Bioinorganic chemistry has modeled important features of this center, aiming at mechanistic understanding and the development of novel catalysts. However, new assays are required for analyzing the effects of ligand variations at the metal ions. By high-resolution X-ray absorption spectroscopy with narrow-band X-ray emission detection (XAS/XES = XAES) and density functional theory (DFT), we studied an asymmetrically coordinated [FeFe] model complex, [(CO)₃Fe^I1-(bdtCl₂)-Fe^I2­(CO)­(Ph₂P–CH₂–NCH₃–CH₂–PPh₂)] (<b>1</b>, bdt = benzene-1,2-dithiolate), in comparison to iron–carbonyl references. Kβ emission spectra (Kβ^1,3, Kβ′) revealed the absence of unpaired spins and the low-spin character for both Fe ions in <b>1</b>. In a series of low-spin iron compounds, the Kβ^1,3 energy did not reflect the formal iron oxidation state, but it decreases with increasing ligand field strength due to shorter iron-ligand bonds, following the spectrochemical series. The intensity of the valence-to-core transitions (Kβ^2,5) decreases for increasing Fe-ligand bond length, certain emission peaks allow counting of Fe-CO bonds, and even molecular orbitals (MOs) located on the metal-bridging bdt group of <b>1</b> contribute to the spectra. As deduced from 3d → 1s emission and 1s → 3d absorption spectra and supported by DFT, the HOMO–LUMO gap of <b>1</b> is about 2.8 eV. Kβ-detected XANES spectra in agreement with DFT revealed considerable electronic asymmetry in <b>1</b>; the energies and occupancies of Fe-d dominated MOs resemble a square-pyramidal Fe(0) for Fe1 and an octahedral Fe­(II) for Fe2. EXAFS spectra for various Kβ emission energies showed considerable site-selectivity; approximate structural parameters similar to the crystal structure could be determined for the two individual iron atoms of <b>1</b> in powder samples. These results suggest that metal site- and spin-selective XAES on [FeFe] hydrogenase protein and active site models may provide a powerful tool to study intermediates under reaction conditions

HOMO and LUMO energies and natural population analysis charges from DFT^a.

<p>HOMO and LUMO energies and natural population analysis charges from DFT<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#t005fn001" target="_blank">^a</a>.</p

Room-Temperature Energy-Sampling Kβ X‑ray Emission Spectroscopy of the Mn₄Ca Complex of Photosynthesis Reveals Three Manganese-Centered Oxidation Steps and Suggests a Coordination Change Prior to O₂ Formation

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn₄Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kβ_1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kβ_1,3 line energies suggest a high-valence configuration of the Mn₄Ca complex with Mn­(III)₃Mn­(IV) in S₀, Mn­(III)₂Mn­(IV)₂ in S₁, Mn­(III)­Mn­(IV)₃ in S₂, and Mn­(IV)₄ in S₃ and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S₄ state unlikely. The difference spectra of both manganese Kβ_1,3 emission and K-edge X-ray absorption display different shapes for Mn­(III) oxidation in the S₂ → S₃ transition when compared to Mn­(III) oxidation in the S₁ → S₂ transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S₂ → S₃ transition, which could be oxidation of five-coordinated Mn­(III) to six-coordinated Mn­(IV). Conceivable options for the rearrangement of (substrate) water species and metal–ligand bonding patterns at the Mn₄Ca complex in the S₂ → S₃ transition are discussed

EXAFS simulation parameters^a.

<p>EXAFS simulation parameters<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#t002fn001" target="_blank">^a</a>.</p

EXAFS spectra of cobalamin systems.

<p>Panel (A) shows Fourier-transforms (FTs) of the EXAFS oscillations in panel (B) for indicated solution Cbl or CoFeSP-Cbl samples. Black lines, experimental data; coloured lines, simulations with parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#pone.0158681.t002" target="_blank">Table 2</a> (fits 2, 5, 7, 10, 12, 14, 16, 19, 21); spectra in (A) and (B) were vertically shifted for comparison.</p

Cobalt-ligand bond lengths from crystallography, EXAFS, and DFT.

<p>Cobalt-ligand bond lengths from crystallography, EXAFS, and DFT.</p