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1. Spectroscopic Characterization of the Divalent Metal Docking Motif to Isolated Cyanobenzoate: Direct Observation of Tridentate Binding to ortho -Cyanobenzoate and Implications for the CN Response

   https://doi.org/10.1021/acs.jpca.2c07658  

   Mohamed, Ahmed; Edington, Sean C.; Secor, Maxim; Breton, James R.; Hammes-Schiffer, Sharon; Johnson, Mark A. (February 2023, The Journal of Physical Chemistry A)

   Cryogenic infrared vibrational spectroscopy of D2-tagged cyanobenzoate (CBA) derivatives are obtained and analyzed to characterize the intrinsic spectroscopic responses of the -CO2‾ head group to its location on the ring in both the isolated anions and the cationic complexes with divalent metal ions, M2+ (M=Mg, Ca, Sr). The benzonitrile functionality establishes the different ring isomers (para, meta, ortho) according to the location of the carboxylate and provides an additional reporter on the molecular response to the proximal charge center. The aromatic carboxylates display slightly smaller shifts than those observed for a related aliphatic system upon metal ion complexation. Although the CBA anions display very similar band patterns for all three ring positions, upon complexation with metal ions, the ortho isomer yields dramatically different spectral responses in both the -CO2‾ moiety and the CN group. This behavior is traced to the emergence of a tridentate binding motif unique to the ortho isomer in which the metal ions bind to both the oxygen atoms of the carboxylate group and the N atom of the cyano group. In that configuration, the -CO2‾ moiety is oriented perpendicular to the phenyl ring, and the CN stretching fundamental is both strong and red-shifted relative to its behavior in the isolated neutral. The behaviors of the metal-bound ortho complexes occur in contrast to the usual blue shifts associated with “Lewis” type binding of metal ions end-on to -CN. The origins of these spectroscopic features are analyzed with the aid of electronic structure calculations, which also explore differences expected for complexation of monovalent cations to the ortho carboxylate. The resulting insights have implications for understanding the balance between electrostatic and steric interactions at metal binding sites in chemical and biological systems. 

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2. Kinetic Characterization and Identification of Key Active Site Residues of the L‐Aspartate N ‐Hydroxylase, CreE

   https://doi.org/10.1002/cbic.202400350  

   Johnson, Sydney_B; Valentino, Hannah; Sobrado, Pablo (July 2024, ChemBioChem)

   Abstract CreE is a flavin‐dependent monooxygenase (FMO) that catalyzes three sequential nitrogen oxidation reactions of L‐aspartate to produce nitrosuccinate, contributing to the biosynthesis of the antimicrobial and antiproliferative nautral product, cremeomycin. This compound contains a highly reactive diazo functional group for which the reaction of CreE is essential to its formation. Nitro and diazo functional groups can serve as potent electrophiles, important in some challenging nucleophilic addition reactions. Formation of these reactive groups positions CreE as a promising candidate for biomedical and synthetic applications. Here, we present the catalytic mechanism of CreE and the identification of active site residues critical to binding L‐aspartate, aiding in future enzyme engineering efforts. Steady‐state analysis demonstrated that CreE is very specific for NADPH over NADH and performs a highly coupled reaction with L‐aspartate. Analysis of the rapid‐reaction kinetics showed that flavin reduction is very fast, along with the formation of the oxygenating species, the C4a−hydroperoxyflavin. The slowest step observed was the dehydration of the flavin. Structural analysis and site‐directed mutagenesis implicated T65, R291, and R440 in the binding L‐aspartate. The data presented describes the catalytic mechanism and the active site architecture of this unique FMO. 

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3. Theory and simulations for RNA folding in mixtures of monovalent and divalent cations

   https://doi.org/10.1073/pnas.1911632116  

   Nguyen, Hung T.; Hori, Naoto; Thirumalai, D. (October 2019, Proceedings of the National Academy of Sciences)

   RNA molecules cannot fold in the absence of counterions. Experiments are typically performed in the presence of monovalent and divalent cations. How to treat the impact of a solution containing a mixture of both ion types on RNA folding has remained a challenging problem for decades. By exploiting the large concentration difference between divalent and monovalent ions used in experiments, we develop a theory based on the reference interaction site model (RISM), which allows us to treat divalent cations explicitly while keeping the implicit screening effect due to monovalent ions. Our theory captures both the inner shell and outer shell coordination of divalent cations to phosphate groups, which we demonstrate is crucial for an accurate calculation of RNA folding thermodynamics. The RISM theory for ion–phosphate interactions when combined with simulations based on a transferable coarse-grained model allows us to predict accurately the folding of several RNA molecules in a mixture containing monovalent and divalent ions. The calculated folding free energies and ion-preferential coefficients for RNA molecules (pseudoknots, a fragment of the rRNA, and the aptamer domain of the adenine riboswitch) are in excellent agreement with experiments over a wide range of monovalent and divalent ion concentrations. Because the theory is general, it can be readily used to investigate ion and sequence effects on DNA properties. 

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4. Computational predictions of metal–macrocycle stability constants require accurate treatments of local solvent and pH effects

   https://doi.org/10.1039/D1CP00611H  

   Gentry, Brian M.; Choi, Tae Hoon; Belfield, William S.; Keith, John A. (April 2021, Physical Chemistry Chemical Physics)

   Rational design of molecular chelating agents requires a detailed understanding of physicochemical ligand–metal interactions in solvent phase. Computational quantum chemistry methods should be able to provide this, but computational reports have shown poor accuracy when determining absolute binding constants for many chelating molecules. To understand why, we compare and benchmark static- and dynamics-based computational procedures for a range of monovalent and divalent cations binding to a conventional cryptand molecule: 2.2.2-cryptand ([2.2.2]). The benchmarking comparison shows that dynamics simulations using standard OPLS-AA classical potentials can reasonably predict binding constants for monovalent cations, but these procedures fail for divalent cations. We also consider computationally efficient static procedure using Kohn–Sham density functional theory (DFT) and cluster-continuum modeling that accounts for local microsolvation and pH effects. This approach accurately predicts binding energies for monovalent and divalent cations with an average error of 3.2 kcal mol −1 compared to experiment. This static procedure thus should be useful for future molecular screening efforts, and high absolute errors in the literature may be due to inadequate modeling of local solvent and pH effects. 

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5. Lanthanide-dependent coordination interactions in lanmodulin: a 2D IR and molecular dynamics simulations study

   https://doi.org/10.1039/D1CP03628A  

   Liu, Stephanie; Featherston, Emily R.; Cotruvo, Joseph A.; Baiz, Carlos R. (October 2021, Physical Chemistry Chemical Physics)

   The biological importance of lanthanides, and the early lanthanides (La 3+ –Nd 3+ ) in particular, has only recently been recognized, and the structural principles underlying selective binding of lanthanide ions in biology are not yet well established. Lanmodulin (LanM) is a novel protein that displays unprecedented affinity and selectivity for lanthanides over most other metal ions, with an uncommon preference for the early lanthanides. Its utilization of EF-hand motifs to bind lanthanides, rather than the Ca 2+ typically recognized by these motifs in other proteins, has led it to be used as a model system to understand selective lanthanide recognition. Two-dimensional infrared (2D IR) spectroscopy combined with molecular dynamics simulations were used to investigate LanM's selectivity mechanisms by characterizing local binding site geometries upon coordination of early and late lanthanides as well as calcium. These studies focused on the protein's uniquely conserved proline residues in the second position of each EF-hand binding loop. We found that these prolines constrain the EF-hands for strong coordination of early lanthanides. Substitution of this proline results in a more flexible binding site to accommodate a larger range of ions but also results in less compact coordination geometries and greater disorder within the binding site. Finally, we identify the conserved glycine in the sixth position of each EF-hand as a mediator of local binding site conformation and global secondary structure. Uncovering fundamental structure–function relationships in LanM informs the development of synthetic biology technologies targeting lanthanides in industrial applications. 

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