Combining Spectroscopy and Theory to Evaluate Structural Models of Metalloenzymes: A Case Study on the Soluble [NiFe] Hydrogenase from Ralstonia eutropha

Hydrogenases catalyse the reversible cleavage of molecular hydrogen into protons and electrons. While most of these enzymes are inhibited under aerobic conditions, some hydrogenases are catalytically active even at ambient oxygen levels. In particular, the soluble [NiFe] hydrogenase from Ralstonia eutropha H16 couples reversible hydrogen cycling to the redox conversion of NAD(H). Its insensitivity towards oxygen has been formerly ascribed to the putative presence of additional cyanide ligands at the active site, which has been, however, discussed controversially. Based on quantum chemical calculations of model compounds, we demonstrate that spectroscopic consequences of the proposed non‐standard set of inorganic ligands are in contradiction to the underlying experimental findings. In this way, the previous model for structure and function of this soluble hydrogenase is disproved on a fundamental level, thereby highlighting the efficiency of computational methods for the evaluation of experimentally derived mechanistic proposals.     

Molecular Recognition of Rosmarinic Acid from Salvia sclareoides Extracts by Acetylcholinesterase: A New Binding Site Detected by NMR Spectroscopy

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer’s disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC‐MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4′‐O‐glucoside, luteolin 3′,7‐di‐O‐glucoside and luteolin 7‐O‐(6′′‐O‐acetylglucoside) were identified by MS² and MS³ data acquired during the LC‐MSⁿ runs. Quantification of rosmarinic acid by HPLC with diode‐array detection (DAD) showed that the butanol extract is the richest one in this component (134 μg mg^?1 extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE‐bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD‐based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.     

Caught in the Hinact: Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O2‐stable State of [FeFe] Hydrogenase

[FeFe] hydrogenases are the most active H₂ converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O₂‐stable state called H_inact. To date, the structure and mechanism of formation of H_inact remain unknown. Our 1.65 Å crystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in‐depth spectroscopic characterization by X‐ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the H_inact state and its mechanism of formation. This may facilitate the design of O₂‐stable hydrogenases and molecular catalysts.