Journal
ACS CATALYSIS
Volume 7, Issue 5, Pages 3569-3574Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.7b00688
Keywords
soybean lipoxygenase; hydrogen tunneling; proton-coupled electron transfer; kinetic isotope effects; nonadiabatic; conformational sampling; protein motions; biocatalysis
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Funding
- National Institute of Health [GM118117-01, GM056207]
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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 (ME) with an average ME 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.
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