Protein-Protein Interaction Surfaces in Diiron Enzymes

University Of Wisconsin-Madison, Madison WI

Investigators

Abstract

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). Intellectual Merit: Protein-protein interactions help to organize complex pathways of intercellular communication, promote efficient handling of essential nutrients within cells, and control the action of enzymes utilized by all living organisms. This project focuses on understanding the essential role of protein-protein interactions in enzyme catalysis. The subject of the research is toluene 4-monooxygenase, a bacterial enzyme complex that plays a prominent role in the catabolism of natural hydrocarbons and man-made pollutants in the environment, and one that is increasingly used for enzymatic synthesis of valuable compounds. Toluene 4-monooxygenase is an iron-containing enzyme. Two iron atoms, called a diiron center, make up the active site where the reaction of electrons, oxygen gas, and toluene occurs. High-resolution crystal structures of the essential T4moH-T4moD complex, acquired during a prior NSF-funded project, suggest a new paradigm for how protein-protein binding will control catalysis. The goal of this research is to experimentally define how protein-protein interactions and their associated structural rearrangements contribute to efficient completion of a highly energetic, multi-step reaction. State-of-the art methods including biochemical kinetics, protein crystallography, metalloprotein spectroscopy, and computer simulations will be employed in the research. Broader Impact: This project is an interdisciplinary effort led by an expert team of internationally recognized researchers with training in biochemistry, biophysics, chemistry, and computational methods. Knowledge gained will define, at the atomic level, how the exquisite interactions of complex protein systems can achieve difficult, highly selective chemical reactions. During the project, a collaborative team of undergraduates, graduate students, and post-graduate fellows will be assembled to complete the scientific objectives. High-school students that successfully complete instruction from the Dane County Youth Apprenticeship Program will be matched into the research laboratory for a 1- or 2-year internship, and will participate in all aspects of the project. The apprentices are nominated by area high schools, and include participants from underrepresented groups, diverse financial backgrounds, students from rural areas, and students that will be the first from their family to attend college. These interactions enhance the diversity of the research environment, increase opportunities for mentoring, and expand the impact of this program to include both research and training of new scientists.