Elucidating the Molecular Mechanism of a Ni Hydrogenase: An Infrared Spectroscopic Approach

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9509064 Bagley This research details the utilization of static and time resolved Fourier transform infrared spectroscopy for delineating the molecular mechanism of hydrogen binding and activation in Ni hydrogenases. The research objectives are: (1) Determination of whether the anomalous infrared bands detected between 2100 and 1900 cm-1 are general features of Ni hydrogenases. To this end a series of infrared studies will be carried out on a well-characterized hydrogenase from Desulfovibrio gigas. (2) Identification of the structure responsible for these unusual infrared signatures using infrared spectroscopy coupled with chemical modification of the enzyme and the study of isotopically labeled enzyme. (3) Utilization of the three infrared signatures for further study of the various redox states of the enzyme. This approach provides a unique opportunity; Ni hydrogenases display at least two EPR-silent species which are distinguishable by their infrared signatures in the 2100 and 1900 cm-1 spectral region. (4) Utilization of mid- infrared spectroscopy to examine the role individual amino acids play in the activation of hydrogen. (5) Examination of the oxidation state of the Ni center in various redox states of the enzymes. This approach used the infrared detectable carbon monoxide stretching frequency, arising from Ni bound carbon monoxide, as a probe of the oxidation state of the Ni. %%% Ni Hydrogenases are enzymes that catalyze the simplest reaction in nature and the simplest conversion of electrical energy into chemical energy of universal utility: 2e- + 2H+ = H2. This research involves a spectroscopic study of the anomalous infrared absorption bands that are characteristic of these enzymes. Infrared spectroscopic studies will be made on the well-characterized Ni hydrogenases from Desulfovibrio gigas. It is a anticipated that this work will have important consequences for understanding the mechanism underlying Ni hydrogenase's ability to split molecular hydrogen into protons and electrons. The work also involves the development of methodologies for handling infrared samples under strictly anaerobic conditions, and should prove of general utility in infrared studies of other oxygen sensitive metallo-proteins. ***