Role of metal coordinated auxiliary residues in catalysis: A structural and mechanistic study.

Metal ions are integral to the structure and function of nearly half of all known proteins, with approximately one-quarter to one-third of proteins directly dependent on these ions for their biological activity. While, catalytic and co-catalytic contributions in them have predominantly been attributed to the metal centre and active site residues, The significance of auxiliary residues coordinated to the metal ion has often been underemphasized, and traditionally viewed as passive elements confined to the role of stabilizing the metal ion. Herein, we present the detailed reaction mechanism of Terephthalate 1,2-cis-dihydrodiol dehydrogenase (TphB), the only known Zinc-dependent oxidative decarboxylase and the role of zinc coordinated histidine residues in catalysis. Over 30 X-ray crystallographic structures of active site variants revealed a notable trend in the Nε-Zn-Nε angles during the course of catalytic transitions. Initially the H159Nε2-Zn-Nε2H203, H255Nε2-Zn-Nε2H203, H159Nε2-Zn-Nε2H255 angles in the native state were 60°, 59.2°, and 55.5°. The angles significantly increased in transition state analogues, by 39.4°, 45.05°, and 42°, then again decrease in the product state by 16.7°, 17.7°, and 17.7°, respectively. This trend aligns with the Jencks "Circe effect," where these angular adjustments introduce a conformational restriction, thereby elevating the system's free energy through entropic loss. These fluxional changes, akin to Berry pseudorotation and the Bartell mechanism, accompanied by continuous cycles of entropic loss and gain, facilitate substrate accommodation, catalytic transitions, transition state stabilization/destabilization, product release, and ultimately, reversion to the enzyme’s initial state. This nuanced understanding could inform the design of effective metal-organic frameworks (MOFs) and (de)carboxylase catalysts with industrial and pharmacological implications, while offering a promising avenue for CO2 capture and plastic upcycling, thereby aligning with the sustainability goals.