Enhanced Rigidification within a Double Mutant of Soybean Lipoxygenase Provides Experimental Support for Vibronically Nonadiabatic Proton-Coupled Electron Transfer Models

• Published in: ACS catalysis Vol. 7; no. 5; pp. 3569 - 3574
• Main Authors: Hu, Shenshen, Soudackov, Alexander V, Hammes-Schiffer, Sharon, Klinman, Judith P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 05.05.2017
• Subjects: Letter; nonadiabatic; hydrogen tunneling; proton-coupled electron transfer; conformational sampling; protein motions; soybean lipoxygenase; kinetic isotope effects; biocatalysis
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.7b00688

Soybean lipoxygenase (SLO) is a prototype for nonadiabatic hydrogen tunneling reactions and, as such, has served as the subject of numerous theoretical studies. In this work, we report a nearly temperature-independent kinetic isotope effect (KIE) with an average KIE value of 661 ± 27 for a double mutant (DM) of SLO at six temperatures. The data are well-reproduced within a vibronically nonadiabatic proton-coupled electron transfer model in which the active site has become rigidified compared to wild-type enzyme and single-site mutants. A combined temperature–pressure perturbation further shows that temperature-dependent global motions within DM-SLO are more resistant to perturbation by elevated pressure. These findings provide strong experimental support for the model of hydrogen tunneling in SLO, where optimization of both local protein and ligand motions and distal conformational rearrangements is a prerequisite for effective proton vibrational wave function overlap between the substrate and the active-site iron cofactor.