This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Atmospheric nitrogen fixation is a tremendous chemical feat accomplished by a few select microorganisms possessing nitrogenase, a unique metalloenzyme. Nitrogenase derives its competency from several iron-sulfur clusters that form a chain of electron transport. Two of these metal clusters, the P-cluster and FeMoco, possess an exceptional structure and composition that make them exciting subjects of study. Little is understood about the biosynthesis of these two complex metalloclusters. Progress made in unraveling the mechanism of their assembly would be invaluable in better understanding not only nitrogen fixation, but also biological metal cluster assembly as a whole. A series of x-ray absorption studies are proposed to ascertain the specific stepwise process through which these clusters are constructed. The first examination will focus on the biosynthesis of P-cluster. In a previous work, a non-native conformation of the Pcluster on a FeMoco-free nitrogenase MoFe protein (designated Av1) was identified. Pre-incubation of this form of MoFe protein at various time lengths with the nifH gene product, the nitrogenase Fe protein (designated Av2), will be performed prior to quench. Determination of the changes in the average iron coordination, oxidation state, and atomic environment by Fe K-edge XAS/EXAFS would render a time-lapse capture of P-cluster assembly and suggest a role of the Fe protein in P-cluster maturation. This will be coupled to concomitant EPR and activity assay analysis, providing a complete picture of P-cluster generation. The second examination will focus on the assembly of FeMoco. A unique assay has been developed that permits a previously unseen transformation of a significant fraction of the iron-only precursor into the mature FeMoco on the NifEN scaffold protein. Determination of the structure of this novel reconstituted precursor by Mo K-edge XAS/EXAFS may identify intermediates in FeMoco construction and establish structural and conditional requisites for FeMoco reactivity.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001209-32
Application #
8362249
Study Section
Special Emphasis Panel (ZRG1-BCMB-P (40))
Project Start
2011-03-01
Project End
2012-02-29
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
32
Fiscal Year
2011
Total Cost
$6,590
Indirect Cost
Name
Stanford University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
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