Monitoring Enzyme Clustering in a Highly Crystalline Metal–Organic Framework by Small‐Angle Neutron Scattering

Abstract

Abstract The molecular‐level investigation of enzyme behavior in confined, cell‐free environments is essential to understanding intrinsic properties and optimizing systems for desired functions. Metal–organic frameworks (MOFs) provide unique structural features that enable the immobilization of bulky biomolecules and allow direct probing of enzymatic behavior under confinement. Here, small‐angle neutron scattering (SANS) was employed to probe porosity changes in a highly crystalline Tb‐mesoMOF and to reveal the spatial arrangement of encapsulated enzymes across long‐range length scales. Structural characteristics such as framework void space were resolved by SANS, while contrast‐matching experiments using D 2 O/H 2 O mixtures suppressed background scattering from the MOF and isolated the enzyme contribution. Compared to unloaded Tb‐mesoMOF, cytochrome c (Cyt. c)‐loaded Tb‐mesoMOF exhibited the emergence of a broad scattering feature at low q (∼0.005 Å −1 ), indicative of enzyme clustering within the framework, accompanied by enhanced loading rate and capacity. Additional structural analyses using complementary techniques further corroborated these findings.