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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 \u223c30 cm\u20131 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 \u223c2-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.<\/jats:p>","DOI":"10.1021\/jacs.6c11881","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T21:48:01Z","timestamp":1788472081000},"page":"38577-38581","source":"Crossref","is-referenced-by-count":0,"title":["Distinct Active-Site\nDynamics of Primitive Catalytic\nAmyloids Revealed by Two-Dimensional Infrared Spectroscopy"],"prefix":"10.1021","volume":"148","author":[{"given":"Zhiyuan","family":"Zhao","sequence":"first","affiliation":[{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qin","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingyao","family":"Wang","sequence":"additional","affiliation":[{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhongneng","family":"Zhou","sequence":"additional","affiliation":[{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9646-9142","authenticated-orcid":true,"given":"Bei","family":"Ding","sequence":"additional","affiliation":[{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Shanghai Jiao Tong University , , ,","place":["Shanghai, China, 200240"]},{"name":"Beijing National Laboratory for Molecular Sciences , ,","place":["Beijing, China, 100190"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,3]]},"reference":[{"issue":"2","key":"2026091609112783400_cit1","doi-asserted-by":"publisher","first-page":"19","DOI":"10.3390\/life7020019","article-title":"Small and Random Peptides: An Unexplored Reservoir of Potentially\nFunctional Primitive Organocatalysts. The Case of Seryl-Histidine","volume":"7","author":"Wieczorek","year":"2017","journal-title":"Life"},{"issue":"187","key":"2026091609112783400_cit2","doi-asserted-by":"publisher","DOI":"10.1098\/rsif.2021.0641","article-title":"Peptides before\nand during the Nucleotide World: An\nOrigins Story Emphasizing Cooperation between Proteins and Nucleic\nAcids","volume":"19","author":"Fried","year":"2022","journal-title":"J. R. Soc. Interface"},{"issue":"5\u20136","key":"2026091609112783400_cit3","doi-asserted-by":"publisher","first-page":"1212","DOI":"10.1016\/j.jinorgbio.2007.12.020","article-title":"Methionine Peptide\nFormation under Primordial Earth Conditions","volume":"102","author":"Li","year":"2008","journal-title":"J. Inorg. Biochem."},{"issue":"45","key":"2026091609112783400_cit4","doi-asserted-by":"publisher","first-page":"9400","DOI":"10.1039\/D2TB01605B","article-title":"Extremophilic\nBehavior of Catalytic Amyloids Sustained by Backbone Structuring","volume":"10","author":"Beasley","year":"2022","journal-title":"J. Mater. Chem. B"},{"issue":"4","key":"2026091609112783400_cit5","doi-asserted-by":"publisher","first-page":"303","DOI":"10.1038\/nchem.1894","article-title":"Short Peptides\nSelf-Assemble to Produce Catalytic Amyloids","volume":"6","author":"Rufo","year":"2014","journal-title":"Nat. Chem."},{"issue":"11","key":"2026091609112783400_cit6","doi-asserted-by":"publisher","first-page":"977","DOI":"10.1038\/s41929-019-0348-x","article-title":"Non-Proteinaceous Hydrolase\nComprised of a Phenylalanine Metallo-Supramolecular Amyloid-like Structure","volume":"2","author":"Makam","year":"2019","journal-title":"Nat. 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Lett."},{"issue":"30","key":"2026091609112783400_cit20","doi-asserted-by":"publisher","first-page":"10773","DOI":"10.1039\/C7NR02675G","article-title":"The Amyloid Architecture Provides\na Scaffold for Enzyme-like Catalysts","volume":"9","author":"Al-Garawi","year":"2017","journal-title":"Nanoscale"},{"issue":"8","key":"2026091609112783400_cit21","doi-asserted-by":"publisher","DOI":"10.1063\/1.1997151","article-title":"Characteristic Two-Dimensional\nIR Spectroscopic Features of Antiparallel and Parallel \u03b2-Sheet\nPolypeptides: Simulation Studies","volume":"123","author":"Hahn","year":"2005","journal-title":"J. Chem. Phys."},{"issue":"3","key":"2026091609112783400_cit22","doi-asserted-by":"publisher","first-page":"432","DOI":"10.1021\/ar700188n","article-title":"Amide I Two-Dimensional\nInfrared Spectroscopy of Proteins","volume":"41","author":"Ganim","year":"2008","journal-title":"Acc. 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Phys."},{"issue":"13","key":"2026091609112783400_cit28","doi-asserted-by":"publisher","first-page":"5511","DOI":"10.1063\/1.477170","article-title":"Vibrational Relaxation\nof Azide Ion in Water: The Role of Intramolecular\nCharge Fluctuation and Solvent-Induced Vibrational Coupling","volume":"109","author":"Morita","year":"1998","journal-title":"J. Chem. Phys."},{"issue":"49","key":"2026091609112783400_cit29","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202211490","article-title":"A Triplet\nLabel Extends Two-Dimensional Infrared Spectroscopy from Pico- to\nMicroseconds","volume":"61","author":"Brunst","year":"2022","journal-title":"Angew. Chem. Int. Ed."},{"issue":"51","key":"2026091609112783400_cit30","doi-asserted-by":"publisher","first-page":"35329","DOI":"10.1021\/jacs.4c13423","article-title":"Revealing Lithium\nIon Transport Mechanisms and Solvation Structures in Carbonate Electrolytes","volume":"146","author":"Pan","year":"2024","journal-title":"J. Am. Chem. Soc."},{"issue":"6","key":"2026091609112783400_cit31","doi-asserted-by":"publisher","DOI":"10.1063\/1.5130940","article-title":"Two-Dimensional\nInfrared Spectroscopic Study of Cytochrome c Peroxidase Activity in\nDeep Eutectic Solvent","volume":"6","author":"Osawa","year":"2019","journal-title":"Struct. Dyn."},{"issue":"12","key":"2026091609112783400_cit32","doi-asserted-by":"publisher","DOI":"10.1063\/1.2772269","article-title":"Frequency-Frequency\nCorrelation Functions and Apodization in Two-Dimensional Infrared\nVibrational Echo Spectroscopy: A New Approach","volume":"127","author":"Kwak","year":"2007","journal-title":"J. Chem. Phys."},{"issue":"20","key":"2026091609112783400_cit33","doi-asserted-by":"publisher","DOI":"10.1063\/1.2927906","article-title":"Taking Apart the Two-Dimensional\nInfrared Vibrational Echo Spectra: More Information and Elimination\nof Distortions","volume":"128","author":"Kwak","year":"2008","journal-title":"J. Chem. Phys."},{"key":"2026091609112783400_cit34","doi-asserted-by":"crossref","DOI":"10.1017\/CBO9780511675935","article-title":"Concepts\nand Methods of 2D\nInfrared Spectroscopy","author":"Hamm","year":"2011"},{"issue":"33","key":"2026091609112783400_cit35","doi-asserted-by":"publisher","first-page":"9775","DOI":"10.1021\/jp406725a","article-title":"An Ion\u2019s Perspective on the\nMolecular Motions of Nanoconfined Water: A Two-Dimensional Infrared\nSpectroscopy Study","volume":"117","author":"Singh","year":"2013","journal-title":"J. Phys. Chem. B"},{"issue":"10","key":"2026091609112783400_cit36","doi-asserted-by":"publisher","first-page":"3528","DOI":"10.1073\/pnas.0508833103","article-title":"Picosecond Dynamics\nof a Membrane Protein Revealed by 2D IR","volume":"103","author":"Mukherjee","year":"2006","journal-title":"Proc.\nNatl. Acad. Sci. U.S.A."},{"issue":"3","key":"2026091609112783400_cit37","doi-asserted-by":"publisher","first-page":"592","DOI":"10.1021\/jp807528q","article-title":"Empirical\nAmide I Vibrational Frequency Map: Application to 2D-IR Line Shapes\nfor Isotope-Edited Membrane Peptide Bundles","volume":"113","author":"Lin","year":"2009","journal-title":"J. Phys. Chem. B"},{"issue":"18","key":"2026091609112783400_cit38","doi-asserted-by":"publisher","first-page":"4247","DOI":"10.1021\/jp993207r","article-title":"Two-Dimensional\nLine Shapes Derived from Coherent Third-Order Nonlinear Spectroscopy","volume":"104","author":"Tokmakoff","year":"2000","journal-title":"J. Phys. Chem. A"},{"issue":"18","key":"2026091609112783400_cit39","doi-asserted-by":"publisher","first-page":"4221","DOI":"10.1016\/S0021-9258(18)95800-X","article-title":"Esterase\nActivities of Human Carbonic\nAnhydrases B and C","volume":"242","author":"Verpoorte","year":"1967","journal-title":"J. Biol. Chem."},{"issue":"23\u201324","key":"2026091609112783400_cit40","doi-asserted-by":"publisher","first-page":"2375","DOI":"10.1002\/anie.199423751","article-title":"The Key\u2013Lock\nTheory and the Induced Fit Theory","volume":"33","author":"Koshland","year":"1995","journal-title":"Angew. Chem.\nInt. Ed. Engl."},{"issue":"6","key":"2026091609112783400_cit41","doi-asserted-by":"publisher","first-page":"438","DOI":"10.1021\/acs.biochem.8b01004","article-title":"A Biophysical\nPerspective on Enzyme Catalysis","volume":"58","author":"Agarwal","year":"2019","journal-title":"Biochemistry"}],"container-title":["Journal of\nthe American\nChemical Society"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/jacs.6c11881","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38577\/68306518\/jacs.6c11881.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jacsat\/article-pdf\/148\/36\/38577\/68306518\/jacs.6c11881.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T13:30:45Z","timestamp":1789565445000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jacsat\/article\/148\/36\/38577\/5415120\/Distinct-Active-Site-Dynamics-of-Primitive"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,3]]},"references-count":41,"journal-issue":{"issue":"36","published-online":{"date-parts":[[2026,9,3]]},"published-print":{"date-parts":[[2026,9,16]]}},"URL":"https:\/\/doi.org\/10.1021\/jacs.6c11881","relation":{},"ISSN":["0002-7863","1520-5126"],"issn-type":[{"value":"0002-7863","type":"print"},{"value":"1520-5126","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,3]]}}}