Detecting and Characterizing the Kinetic Activation of Thermal Networks in Proteins: Thermal Transfer from a Distal, Solvent-Exposed Loop to the Active Site in Soybean Lipoxygenase

The rate-limiting chemical reaction catalyzed by soybean lipoxygenase (SLO) involves quantum mechanical tunneling of a hydrogen atom from substrate to its active site ferric-hydroxide cofactor. SLO has emerged as a prototypical system for linking the thermal activation of a protein scaffold to the efficiency of active site chemistry. Significantly, hydrogen–deuterium exchange-mass spectrometry (HDX-MS) experiments on wild type and mutant forms of SLO have uncovered trends in the enthalpic barriers for HDX within a solvent-exposed loop (positions 317–334) that correlate well with trends in the corresponding enthalpic barriers for kcat. A model for this behavior posits that collisions between water and loop 317–334 initiate thermal activation at the protein surface that is then propagated 15–34 A inward toward the reactive carbon of substrate in proximity to the iron catalyst. In this study, we have prepared protein samples containing cysteine residues either at the tip of the loop 317–334 (Q322C) or on a c...