Role of Conformational Dynamics in the Evolution of Retro-Aldolase Activity

Enzymes exist as ensembles of conformations that are important for function. Tuning these populations of conformational states through mutation enables evolution toward additional activities. Here we computationally evaluate the population shifts induced by distal and active site mutations in a family of computationally designed and experimentally optimized retro-aldolases. The conformational landscape of these enzymes was significantly altered during evolution, as pre-existing catalytically active conformational substates became major states in the most evolved variants. We further demonstrate that key residues responsible for these substate conversions can be predicted computationally. Significantly, the identified residues coincide with those positions mutated in the laboratory evolution experiments. This study establishes that distal mutations that affect enzyme catalytic activity can be predicted computationally and thus provides the enzyme (re)­design field with a rational strategy to determine promising sites for enhancing activity through mutation

The Frozen Cage Model: A Computationally Low-Cost Tool for Predicting the Exohedral Regioselectivity of Cycloaddition Reactions Involving Endohedral Metallofullerenes

Functionalization of endohedral metallofullerenes (EMFs) is an active line of research that is important for obtaining nanomaterials with unique properties that might be used in a variety of fields, ranging from molecular electronics to biomedical applications. Such functionalization is commonly achieved by means of cycloaddition reactions. The scarcity of both experimental and theoretical studies analyzing the exohedral regioselectivity of cycloaddition reactions involving EMFs translates into a poor understanding of the EMF reactivity. From a theoretical point of view, the main obstacle is the high computational cost associated with this kind of studies. To alleviate the situation, we propose an approach named the frozen cage model (FCM) based on single point energy calculations at the optimized geometries of the empty cage products. The FCM represents a fast and computationally inexpensive way to perform accurate qualitative predictions of the exohedral regioselectivity of cycloaddition reactions in EMFs. Analysis of the Dimroth approximation, the activation strain or distortion/interaction model, and the noncluster energies in the Diels–Alder cycloaddition of <i>s-cis</i>-1,3-butadiene to X@<i>D</i>_3<i>h</i>-C₇₈ (X = Ti₂C₂, Sc₃N, and Y₃N) EMFs provides a justification of the method

Reactivity of an Fe^IV-Oxo Complex with Protons and Oxidants

High-valent Fe-OH species are often invoked as key intermediates but have only been observed in Compound II of cytochrome P450s. To further address the properties of non-heme Fe^IV-OH complexes, we demonstrate the reversible protonation of a synthetic Fe^IV-oxo species containing a tris-urea tripodal ligand. The same protonated Fe^IV-oxo species can be prepared via oxidation, suggesting that a putative Fe^V-oxo species was initially generated. Computational, Mössbauer, XAS, and NRVS studies indicate that protonation of the Fe^IV-oxo complex most likely occurs on the tripodal ligand, which undergoes a structural change that results in the formation of a new intramolecular H-bond with the oxido ligand that aids in stabilizing the protonated adduct. We suggest that similar protonated high-valent Fe-oxo species may occur in the active sites of proteins. This finding further argues for caution when assigning unverified high-valent Fe-OH species to mechanisms