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>

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

Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus

Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (<i>Rc</i>FDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO^–) to CO₂ oxidation. We characterized the molecular structure of the active site of wildtype <i>Rc</i>FDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S­(Cys386)) to Mo­(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo­(V) in a chemically reduced enzyme, and replacement of only the S­(Cys386) ligand by an oxygen of formate upon Mo­(IV) formation. The lack of a Mo=S bond in <i>Rc</i>FDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo–S­(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Mo–ligands are essential for catalysis. Low-pH inhibition of <i>Rc</i>FDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S­(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of <i>Rc</i>FDH