Distinct Active-Site Dynamics of Primitive Catalytic Amyloids Revealed by Two-Dimensional Infrared Spectroscopy

Catalytic amyloids are proposed evolutionary precursors of modern enzymes, yet the active-site dynamics underlying their distinct catalytic properties remain unclear. Here, we combine azide infrared probes and two-dimensional infrared (2D-IR) spectroscopy to characterize the active-site dynamics of primitive catalytic amyloids and compare them with human carbonic anhydrase II (CAII), a representative modern enzyme. The Zn2+-bound azide in amyloids exhibits a ∼30 cm-1 blue shift and a 2-fold longer vibrational lifetime than free azide, establishing vibrational lifetime as a sensitive reporter of metal coordination. Center Line Slope (CLS) analysis reveals the absence of picosecond dynamics in bound azide, indicating that rigid amyloid scaffolds lack the rapid hydration and side-chain motions present in CAII. Furthermore, 2D-IR linewidth analysis shows that amyloids possess ∼2-fold greater inhomogeneous broadening. Such pronounced active-site heterogeneity correlates with reduced catalytic efficiency in amyloids, whereas CAII achieves high activity through a homogeneous and dynamically optimized active site. This contrast reveals two evolutionarily divergent catalytic strategies: conformational selection versus tuned dynamics.