Crystallographic Studies of Nitrogenase

Author: Maggiolo, Ailiena Okumura

Year: 2023

Degree: Dissertation (Ph.D.)

Advisor: Rees, Douglas C.

Committee Members: Clemons, William M.; Gray, Harry B.; Bjorkman, Pamela J.; Rees, Douglas C.

Option: Chemistry

DOI: 10.7907/p232-4w49

Abstract

Nitrogenase is the only enzyme known to reduce atmospheric dinitrogen to ammonia, producing a biologically available form of nitrogen. The primary component of nitrogenase, the molybdenum-iron (MoFe) protein, binds and turns over substrate after reduction by multiple electron equivalents, which are supplied by the obligate reductase, the iron (Fe) protein. Previous high-resolution X-ray crystal structures have provided pictures of the nitrogenase proteins and revealed the geometry of their metalloclusters. In this thesis, we study MoFe protein crystal isoforms and their crystal pathologies to classify isomorphous candidates suitable for crystallographic merging. We then leverage this classification to determine a high-resolution structure of the MoFe protein with improved geometric accuracy and lower coordinate error than currently available models. The reduced states of the MoFe protein are challenging to capture and therefore have been minimally characterized. We explore the structural consequences of introducing reductants and oxidants into MoFe protein crystals and study the effects of X-ray induced photoreduction on the metalloclusters. Further, we determine the radiation damage-free X-ray crystal structure of MoFe protein. The Fe protein adopts various conformational states as it functions in ATP-coupled electron transfer to the MoFe protein. We examine a set of proteins that are evolutionarily related to the Fe protein and have diverse functionalities, but retain similarity in their ATP-dependent function and allostery as a result of their conserved structural features. Finally, we characterize the structural and functional aspects of the Fe protein lacking an iron-sulfur cluster. These studies expand our understanding of the structural properties of nitrogenase and shed light on previously uncharacterized states of these proteins.

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