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

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 D₂O/H₂O 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.