Nitrogenase consists of two metalloproteins, the MoFe protein and the Fe protein. The MoFe protein is an α2β2 heterotetramer and the Fe protein is an α2 homodimer. The catalytic cycle of nitrogenase involves binding of the Fe protein to each αβ catalytic half of the MoFe protein, electron transfer followed by ATP hydrolysis, Pi release and eventually dissociation of the two proteins. This cycle has to be repeated eight consecutive times to reduce one molecule of N2.
The two catalytic halves of the MoFe protein had been considered to be independent of each other. The research presented here showed that there is negative cooperativity associated between the two catalytic halves of the MoFe protein. The results suggested that only one half of the MoFe protein is operative during the first turnover of the enzyme.
In order to understand the substrate reduction mechanism of nitrogenase, the study focused on two important enzymes of the biogeochemical nitrogen cycle: nitrite (NO2 -) and nitrate (NO3 -). Two intermediates of NO2 - reduction were trapped by a remodeled nitrogenase (α-70Ala/α-195Gln MoFe protein) and characterized by advanced spectroscopic studies. These intermediates were found to be identical to the intermediates trapped during reduction of diazene (N2H2) and hydrazine (N2H4). The pathway for reduction NO2 - to ammonia (NH3) was also proposed.
NO3 - was established as a new substrate of nitrogenase. The advanced spectroscopic studies confirmed that the same two intermediates were trapped by the remodeled nitrogenase. Kinetic studies showed that two competing pathways lead to NO3 - reduction by nitrogenase, a primary 2 e- reduction pathway to form nitrite and a secondary 8 e- reduction pathway to form NH3. The pathways for reduction of NO3 - to NO2 - and NH3 were proposed.
| Identifer | oai:union.ndltd.org:UTAHS/oai:digitalcommons.usu.edu:etd-6824 |
| Date | 01 May 2017 |
| Creators | Shaw, Sudipta |
| Publisher | DigitalCommons@USU |
| Source Sets | Utah State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | All Graduate Theses and Dissertations |
| Rights | Copyright for this work is held by the author. Transmission or reproduction of materials protected by copyright beyond that allowed by fair use requires the written permission of the copyright owners. Works not in the public domain cannot be commercially exploited without permission of the copyright owner. Responsibility for any use rests exclusively with the user. For more information contact digitalcommons@usu.edu. |
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