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Crystallographic Studies of Nitrogenase


Maggiolo, Ailiena Okumura (2023) Crystallographic Studies of Nitrogenase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/p232-4w49.


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.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:nitrogenase; nitrogen fixation, X-ray crystallography
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Rees, Douglas C.
Thesis Committee:
  • Clemons, William M. (chair)
  • Gray, Harry B.
  • Bjorkman, Pamela J.
  • Rees, Douglas C.
Defense Date:24 May 2023
Record Number:CaltechTHESIS:06062023-051752771
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for Chapter 6
Maggiolo, Ailiena Okumura0000-0003-1707-5060
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:16088
Deposited By: Ailiena Maggiolo
Deposited On:07 Jun 2023 15:14
Last Modified:08 Nov 2023 00:23

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