Mechanisms of Action in Engineered Isoforms of the Diiron Toluene-4-Monooxygenase Complex

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

Toluene 4-monooxygenase (T4MO) enzyme complex in naturally occurring microbes participates in the metabolism of toluene and can also catalyze the degradation of priority pollutants such as trichloroethylene and chloroform. The natural diiron enzyme complex and highly active mutated isoforms created in previous project period will be studied. The overall objective of this research is to increase our understanding of oxidation catalysis, with a specific focus on determining the origin of high regiospecificity observed in a physiologically relevant aromatic hydroxylation reaction. The experimental goals are to determine the interplay between substrate, active site, and oxidant leading to this highly desirable catalytic outcome. Steady-state and transient kinetic methods will be used to determine intramolecular isotope effects and deuterium shift/retention patterns in studies of the mechanism of aromatic hydroxylation. Alkane "radical clocks" will be studied in combination with 18O2 and 18OH2 labeling of products to study the mechanism of alkane hydroxylation. Rapid-mix/quench methods will be used to study fast reaction intermediates. Multinuclear, multidimensional NMR methods will be used to study protein interactions. Broader Impacts: The project will be undertaken as an interdisciplinary effort involving personnel with advanced training in biochemistry, biophysics, and chemistry. Collaborative interactions involve researchers at both the graduate and undergraduate institutions. These interactions enhance the vigor of the research and teaching efforts of all participants, increase opportunities for diversified training of individuals at each institution, and expand the impact of this program to a national scope. The training of postdoctoral research associates and graduate research assistants will be an integral aspect in the execution of the proposed research. Moreover, this will continue a well-established tradition of the involvement of undergraduates, women, and members of underrepresented groups.