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Analytical Chemistry Investigations Toward Understanding the Mechanism of Nitrogenase from Azotobacter vinelandii and the Role of the 4Fe-4S Cluster of Dna2 from Saccharomyces cerevisiae

Citation

MacArdle, Siobhán Gaustad (2022) Analytical Chemistry Investigations Toward Understanding the Mechanism of Nitrogenase from Azotobacter vinelandii and the Role of the 4Fe-4S Cluster of Dna2 from Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/afrw-sx78. https://resolver.caltech.edu/CaltechTHESIS:05252022-173757484

Abstract

Iron sulfur clusters are ubiquitous metal cofactors that play a variety of roles in many enzymes important for health and the climate. The bacterial nitrogenase enzyme, which supports the growth of all organisms by converting atmospheric dinitrogen into ammonia, contains three different redox-active iron sulfur clusters that are central to its function. Dna2, found in all eukaryotes, is integral to genome maintenance and coordinates an iron sulfur cluster of unknown function. Many details of the nitrogenase mechanism are yet to be revealed and pursuits toward this goal will support human efforts to develop more sustainable solutions to nitrogen fixation, which is required for maintaining our food supply. Thorough characterization of the DNA-maintenance enzyme Dna2 will allow us to develop better technologies for cancer prevention and treatment. Development and optimization, as well as technical critique, of a variety of analytical chemistry techniques were performed toward the goal of increasing our understanding of these two important enzymes. Yeast Dna2 was successfully overexpressed and purified from E. coli and spectroscopic features of the 4Fe-4S cluster were characterized. Toward measuring the redox potential of the 4Fe-4S cluster of Dna2, the DNA-modified electrochemistry technique was evaluated leading to the discovery that the source of electrochemical signals proposed to be due to redox activity of 4Fe-4S clusters in DNA-binding proteins are actually due to the redox activity of Fe-EDTA complexes that form in the buffers of these proteins. These results will support future scientists in accurately interpreting the electrochemical signals from DNA-modified electrochemistry. The solvent isotope effect of nitrogenase reduction was investigated by measuring deuterium incorporation into nitrogenase products by GC-MS, FTIR and NMR, revealing that the enzyme exhibits modest preference for H vs. D in acetylene reduction to ethylene, but significant preference for H in the reduction of protons to dihydrogen. These results indicate that there are distinct mechanisms of H atom transfer in the reduction of these two substrates and the experimental design that we developed opens the door for a new avenue of nitrogenase research to reveal the solvent isotope effects of reduction of a variety of different substrates under different experimental conditions. Finally, a new ATPase assay using ion chromatography was developed to measure ATPase activity of the Fe protein, which provides a tool for future pursuits toward quantifying inorganic phosphate release by ATPases and led to our surprising result that the apo-form of the Fe protein is active in ATP hydrolysis.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:nitrogenase, iron sulfur cluster enzymes, isotope effects, protein electrochemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Rees, Douglas C.
Thesis Committee:
  • Gray, Harry B. (chair)
  • Agapie, Theodor
  • Campbell, Judith L.
  • Rees, Douglas C.
Defense Date:12 May 2022
Funders:
Funding AgencyGrant Number
HHMIUNSPECIFIED
NIHUNSPECIFIED
Parsons FellowshipUNSPECIFIED
Grace Potter Rice FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:05252022-173757484
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:05252022-173757484
DOI:10.7907/afrw-sx78
ORCID:
AuthorORCID
MacArdle, Siobhán Gaustad0000-0001-7843-5977
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14619
Collection:CaltechTHESIS
Deposited By: Siobhan MacArdle
Deposited On:02 Jun 2022 19:57
Last Modified:08 Jun 2022 22:43

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