Structural and Biochemical Characterization of Ligand Bound States of the FeMo-Cofactor of Nitrogenase

Author: Perez, Kathryn A.

Year: 2016

Degree: Dissertation (Ph.D.)

Advisor: Rees, Douglas C.

Committee Members: Beauchamp, Jesse L.; Okumura, Mitchio; Cai, Long; Rees, Douglas C.

Option: Chemistry

DOI: 10.7907/Z9M043DX

Abstract

Nitrogenase is the only known enzyme capable of nitrogen fixation, the reduction of dinitrogen to ammonia, a metabolically available form of nitrogen. Developing an understanding of the complex mechanism required for biological nitrogen fixation requires that the enzyme be characterized in catalytically relevant states, such as those involving ligand binding and reduction. Nitrogenase catalyzes this reaction through the cyclic interaction of two metalloproteins, the Fe-protein and the MoFe-protein which contain three distinct metalloclusters, in an ATP-hydrolysis dependent electron transfer reaction. The binding and subsequent reduction of substrates requires multiple electrons donated from the Fe-protein to the MoFe-protein, in which the active site is located. In this study, we have structurally characterized the binding of two inhibitors to the FeMo-cofactor, CO and the Se of SeCN-. Both interactions involve the displacement of a single S, and the Se was used as a label to follow the interchange of three S sites within the FeMo-cofactor during catalysis. These finding change any future approaches to characterize the mechanism of biological nitrogen fixation, requiring that structural changes be considered for substrate binding and reduction.

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