November 30, 2009 Department Of Chemistry University Of Wisconsin

November 30, 2009 Department of Chemistry University of Wisconsin - Madison Dear Search Committee Members: Robert Usselman worked as a postdoctoral research associate in our laboratory from April 2006 to January 2008 on a collaborative project with Frank Frerman at the University of Colorado Health Sciences Center to determine the electron transfer pathway in electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO). Before he arrived, we had developed electron paramagnetic resonance (EPR) methods to characterize the paramagnetic ironsulfur cluster and the native semiquinone and to measure the distance between the two paramagnetic centers. The plan was to introduce mutations in the vicinity of the paramagnetic cofactors and determine the changes in redox potentials and corresponding changes in enzymatic activity. At the time Robert joined the group the molecular biology to generate mutations had been worked out. However none of us had experience in protein electrochemistry and protein titration curves. We had expected that a collaborator at another University would do that part of the work, but it became evident that the effort required was more than that person could allocate to the task. Although Robert had no prior experience in electrochemistry, he cheerfully offered to learn how to do it. With little assistance from me or from Frank, Robert took the initiative to read the literature, contact other people for advice, and teach himself how to do the titrations and follow the titrations by EPR. The EPR detection of the paramagnetic species is much more specific than the optical spectroscopy that had been used in the past for these systems. As a result of this work he is the first author on a paper describing mutants in the vicinity of the iron-sulfur cluster and the co-author on two related papers. Robert has excellent ‘hands’ in the lab. He helped with the protein preparation and purification, designed and performed the electrochemistry experiments, did pulsed and CW EPR experiments on a Bruker E580, and analyzed the experimental data. He is an excellent experimentalist. Robert’s presentations at our weekly research group meetings were clear and informative. He is an excellent candidate for a faculty position. Sincerely,

Sandra S. Eaton

John Evans Professor and Department Chair, 303-871-3102