Temporal and spatial resolution of distal protein motions that activate hydrogen tunneling in soybean lipoxygenase

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 define a catalytically-linked, radiating cone of aliphatic side chains that connects an active site iron center of SLO to the protein-solvent interface. Employing eight variants of SLO that have been appended with a fluorescent probe at the identified surface loop, nano-second fluorescence Stokes shifts have been measured. We report a remarkable identity of the energies of activation (Ea) for the Stokes shifts decay rates and the millisecond C-H bond cleavage step that is restricted to side chain mutants within an identified thermal network. These findings implicate a direct coupling of distal protein motions surrounding the exposed fluorescent probe to active site motions controlling catalysis. While the role of dynamics in enzyme function has been predominantly attributed to a distributed protein conformational landscape, the presented data implicate a thermally initiated, cooperative protein reorganization that occurs on a timescale faster than nano-second and represents the enthalpic barrier to the reaction of SLO.

Automated summary

The study investigates the deep tunneling mechanisms in hydrogen transfer catalysis using soybean lipoxygenase (SLO) as a prototype enzyme. Through X-ray studies and hydrogen-deuterium exchange experiments, a radiating cone of aliphatic side chains connecting the active site iron center of SLO to the protein-solvent interface is identified. The findings suggest a direct coupling between distal protein motions and active site motions in controlling catalysis.