Sponsoring Institution
National Institute of Food and Agriculture
Project Status
Funding Source
Reporting Frequency
Accession No.
Grant No.
Project No.
Proposal No.
Multistate No.
Program Code
Project Start Date
Aug 1, 2003
Project End Date
Jul 31, 2006
Grant Year
Project Director
Hales, B. J.
Recipient Organization
Performing Department
Non Technical Summary
Nitrogen fixation, the conversion of dinitrogen from the air into ammonia, is directly related to world-wide crop production and, as such, is one of the most important enzymatic reactions in nature. The nitrogen-fixing enzyme, nitrogenase, is synthesized in a relatively small number of diverse soil-living bacteria as well as photosynthetic bacteria and cyanobacteria. Of all the ammonia present in the world, well over 60% has been generated from bacterial nitrogen fixation. Obviously, it would be of great importance to agricultural yield to have a clear understanding of the mechanism of this system. In spite of the large volume of research that has been done on nitrogenase, the mechanism of nitrogen fixation and the binding mode needed for substrate reduction is still unclear. The purpose of this research project is to gain a better understanding of this mechanism by using EPR, MCD and ENDOR spectroscopic techniques to study intermediate states of both normal and mutant forms of the Mo-nitrogenase enzyme. These techniques will be used to detect and elucidate the mode of binding as well as the binding sites of the substrate acetylene as well as the inhibitor CO. The variaous oxidation states of the enzyme's other metal cluster, the P cluster, will also be investiated. The results of these experiments will provide very important information to further our understanding of the basic enzymatic process of nitrogenase.
Animal Health Component
Research Effort Categories

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
2000 - Chemistry;
Goals / Objectives
During the two year period of the grant, (1) the CO-induced EPR signals will be investigation using variant MoFe proteins containing amino acid substitutions in the region of the FeMo cofactor, (2) an MCD study will be undertaken of the MoFe protein in the S1 state, (3) an MCD study will be undertaken of the photo-conversion of hi-CO into lo-CO, (4) 57Fe ENDOR study will be conducted on acetylene bound to the FeMo cofactor, and (5) an EPR/MCD study will be undertaken on the P+ state of both the DnifH apo-MoFe protein and the DnifB apo-MoFe protein.
Project Methods
In all of the studies, EPR, MCD and ENDOR spectroscopic techniques will be used. All of these techniques have the advantage that they probe only the protein's paramagnetic sites. Proteins will be purified by conventional methods and turnover samples will be prepared in the presence of either the inhibitor CO or the substrate, acetylene. Both of these molecules have been shown to elicit intense EPR signals corresponding to the molecules bound to the enzyme's active site, FeMo-co. MCD has not previously been used to intestigate the nitrogenase P clusters. Our study will help monitor structural changes in this cluster during oxidation.

Progress 10/01/04 to 09/30/05

During the past year our group has been very active in understanding the mechanism of nitrogen fixation. We were able to show that different variant enzymes change there ability to be inhibited by CO by simple changes in the amino acids around the FeMo cofactor. This study gives us a strong indication of the regions of the active-site cofactor where substrates and inhibitor bind and how they bind. We have also, for the first time, been able to show the reduction state needed for initial substrate binding prior to reduction. This state occurs two electrons more reduced that the normal as-isolated state of the enzyme. This state, to date, cannot be achieved other than by enzymatic reduction. Therefore, the enzyme must undergo two electron transfer cycles prior to substrate binding.

The resutls from these studies greatly advance our understanding of the mechanism of nitrogen fixation and, with them, add to our abiliy to model nitrogen fixing metal clusters for subsequent non-enzymatic reduction reactions of dinitrogen and similar multipe bonded molecules.


  • Variant MoFe Proteins of Azotobacter vinelandii: Effects of Carbon Monoxide on Electron Paramagnetic Resonance Spectra Generated During Enzyme Turnover, Zofia Maskos, Karl Fisher, Morten Sorlie, William Newton and Brian Hales, (2005) J. Biol. Inorg. Chem., 10, 394-406.
  • Electron Inventory, Kinetic Assignment (En), Structure, and Bonding of Nitrogenase Turnover Intermediates with C2H2 and CO, Hong-In Lee, Morten Sorlie, Jason Christiansen, Tran-Chin Yang, Junlong Shao, Dennis R. Dean, Brian J. Hales, and Brian Hoffman (2005) J. Amer. Chem. Soc., 127, 15880-15890.