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

Proc Natl Acad Sci U S A. 2023 Mar 7;120(10):e2211630120. doi: 10.1073/pnas.2211630120. Epub 2023 Mar 3.

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 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, nanosecond 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 nanosecond and represents the enthalpic barrier to the reaction of SLO.

Keywords: Stokes shift decay; hydrogen tunneling; hydrogen–deuterium exchange; room temperature X-ray; thermal activation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Fluorescent Dyes
  • Glycine max*
  • Hydrogen
  • Lipoxygenase*
  • Motion

Substances

  • Lipoxygenase
  • Fluorescent Dyes
  • Hydrogen