Impact of mutation on the proton-coupled electron transfer reaction catalyzed by soybean lipoxygenase

Open Access
Author:
Edwards, Sarah J
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
April 19, 2010
Committee Members:
  • Sharon Hammes Schiffer, Dissertation Advisor
  • Sharon Hammes Schiffer, Committee Chair
  • James Bernhard Anderson, Committee Member
  • Albert Welford Castleman Jr., Committee Member
  • John H Golbeck, Committee Member
Keywords:
  • soybean lipoxygenase
  • proton-coupled electron transfer
  • kinetic isotope effects
  • driving foce dependence
  • rate constant expressions
  • mutant enzymes
Abstract:
The hydrogen atom abstraction catalyzed by soybean lipoxygenase occurs by way of a proton- coupled electron transfer mechanism. Experimental kinetic studies indicate that the deuterium kinetic isotope effect for the reaction is ~80 at room temperature and exhibits weak temperature dependence. Kinetic studies also show that mutation of the I553 residue in lipoxygenase, which is ~15 Å from the active site Fe but borders the substrate, can significantly alter the magnitude and temperature dependence of the kinetic isotope effect for this reaction. We have studied the wild-type and mutant lipoxygenase with a vibronically nonadiabatic theory of proton coupled- electron transfer that includes the quantum effects of the electrons and transferring proton. This theory indicates that the magnitude and temperature dependence of the kinetic isotope effect depend on the proton donor-acceptor equilibrium distance and vibrational frequency, as well as the overlap between the reactant and product proton vibrational wavefunctions. We used this theoretical treatment to analyze the experimental data for the wild-type and four I553 mutant lipoxygenases. We found that the proton donor-acceptor equilibrium distance increases and the vibrational frequency decreases as the residue I553 becomes less bulky. These changes at the proton transfer interface are correlated to the experimentally observed increase in both the magnitude and temperature dependence of the KIE as the residue I553 becomes less bulky. We performed all-atom molecular dynamics simulations for the entire solvated lipoxygenase enzyme, as well as two I553 mutants, in order to understand how a mutation at residue I553 impacts the proton transfer interface. These simulations provided insight into the impact of mutation on the substrate mobility, conformation and orientation.