Date of Award:

2016

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Chemistry and Biochemistry

Advisor/Chair:

Lance C. Seefeldt

Abstract

Nitrogenase, EC: 1.18.6.1 is the enzyme that catalyzes the reduction of dinitrogen to ammonia; this is known as biological nitrogen fixation. Nitrogen fixation is so important to our daily lives, that we utilize approximately 2% of the annual energy produced worldwide to fix nitrogen industrially via the Haber-Bosch process. The industrial process requires a high input of energy in the form of heat (>450°C) and pressure (>200 atm>), while the enzymatic system is performed under ambient conditions. Research invested into understanding the mechanism of this biological catalyst could eventually lead to understanding how nature performs difficult chemical reductions, which could allow researchers to develop catalysts that mimic this enzyme to perform many important reactions, such as nitrogen fixation, much more efficiently than today.

Electron transfer in the nitrogenase is only partially understood, and is one of the key elements of understanding the mechanism of nitrogenase. Nitrogenase is composed of two proteins, the Fe protein delivers electrons to the MoFe protein, where N2 binds and is subsequently reduced. The conformational changes that take place upon Fe protein binding were investigated in order to better understand electron transfer within the enzyme. Further, studies were performed which probed the P-cluster, an iron sulfur cluster in the MoFe protein that acts as an intermediate in the electron transfer event, and successfully identified the biologically relevant redox state of the P-cluster, P+1. Other studies were performed which identified several variants of the MoFe protein which were able to accept electrons from a chemical reductant. These variants are the first examples of nitrogenase enzymes able to accept electrons from any source other than Fe protein and shown substrate reduction. These variants pinpoint where nitrogenase is likely to undergo conformational changes to allow electron transfer to the active site of the enzyme. Finally, studies were done on the isolated active site of the protein, the iron molybdenum cofactor to better understand how the active site of nitrogenase works The goal of this thesis is to better understand how electrons travel through nitrogenase, and how they are utilized at the active site, FeMo-cofactor, when they arrive.

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