RUI: Infrared Spectroscopy as a Probe of Active Site Structure and Molecular Mechanism in the Hydrogenases

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In this project infrared difference spectroscopy will be used to study the active site structure and molecular mechanism of hydrogen binding and activation in the hydrogenases. Previous infrared spectroscopic studies have shown that both the [NiFe] hydrogenases and the Fe-only hydrogenases display intense infrared bands in the 2100-1800 cm-1 spectral region. The groups responsible for these infrared bands have been identified as arising from intrinsic carbon monoxide and cyanide molecules coordinated to Fe ions in the active site of these enzymes. The primary objectives of this research are: 1) Utilization of infrared spectroscopy for delineating the active site structure of the [NiFe] hydrogenase from Desulfovibrio vulgaris. The enzyme from Desulfovibrio vulgaris has been proposed to have a NiFe (CO)(CN)(SO) cluster at its active site rather than the NiFe (CO)(CN)2 cluster shown to exist in other [NiFe] Hydrogenases. 2) Utilization of infrared difference spectroscopy to explore the molecular changes accompanying reductive activation in the [NiFe] hydrogenases. 3) Utilization of the infrared spectral signatures, combined with isotopic labeling, for determining the active site structure of Fe-only Hydrogenases I and II from Clostridium pasteurianum. 4) Utilization of mid-infrared difference spectroscopy to examine the role amino acids play in the activation of hydrogen. The research will involve undergraduate students pursuing degrees in chemistry and chemistry-related fields.It is anticipated that this work will have important consequences for understanding the mechanism underlying the hydrogenases' ability to split molecular hydrogen into protons and electrons, or conversely, to produce hydrogen from protons and electrons. Such knowledge has consequences for the design of cheap catalysts for the production of hydrogen, a clean burning fuel source. The proposed research will involve the participation of undergraduate students, both during the academic year and during the summer. Student participation in this project will afford undergraduate students a unique opportunity to apply skills and knowledge they have learned in their coursework, while contributing to an exciting project in modern physical biochemistry.