Temporal and Spatial Resolution of a Protein Quake that Activates Hydrogen Tunneling in Soybean Lipoxygenase

Abstract

The enzyme soybean lipoxygenase (SLO) provides a prototype for deep tunneling mechanisms in hydrogen transfer catalysis. This work combines room temperature X-ray studies with extended hydrogen deuterium exchange experiments to detect a radiating cone of aliphatic side chains that extends from the iron active site of SLO to the protein-solvent interface. Employing eight variants of SLO, nanosecond fluorescence Stokes shifts have been measured using a probe appended to the identified surface loop. We report a remarkable identity of the enthalpies of activation for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within the identified thermal network. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, these new data implicate a thermally initiated, cooperative protein quake as the source of the activation of SLO. These findings indicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis.