Title page for ETD etd-04132004-141920

Type of Document Dissertation
Author Broach, Robyn B
Author's Email Address rbroac1@lsu.edu
URN etd-04132004-141920
Title Spectroscopic Investigation of Nitrogenase: EPR and MCD Studies of the FeMo Cofactor and the P-Cluster
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Brian J. Hales Committee Chair
Erwin Poliakoff Committee Member
George Stanley Committee Member
Vincent LiCata Committee Member
Arthur M. Sterling Dean's Representative
  • mcd spectroscopy
  • femo cofactor
  • p-cluster
  • nitrogenase
  • epr spectroscopy
Date of Defense 2004-03-31
Availability unrestricted
Little is known about substrate binding and reduction of nitrogenase. EPR spectroscopy is used here to observe intermediate states generated by different substrates. Two different spin states (S=3/2 and S=1/2) were exhibited for each substrate, which may result from different binding of the substrate to the cofactor (side-on or terminal binding) or the difference of the substrate binding to either Fe or Mo of the cofactor. Parallel studies were performed on a variant MoFe protein, alpha-195Gln, which exhibited different signals from the wild-type suggesting that the substituted amino acid maybe necessary to reach some mechanistic states that the wild-type MoFe protein can reach.

Electron transfer between the Fe protein and the MoFe protein was investigated to help determine the initial electron transfer pathway in nitrogenase. The altered Fe protein, L127-deletion Fe protein, is permanently in the complex-ready conformation and complexes with the MoFe protein to allow one electron transfer. The MCD studies suggest the presence of a second paramagnetic center in addition to the resting state cofactor. The second paramagnetic center may result from an electron delocalized over the entire P-cluster or its return to the Fe protein.

The P-cluster is suggested to play a role in the electron transfer from the Fe protein to the cofactor. Apo-proteins were used to provide information about the function and the maturation of the P-cluster. One apo-protein, nifB-deletion MoFe protein, exhibits redox characteristics analogous to the wild-type MoFe protein, i.e., both as-isolated proteins have the P-cluster in the state P0 and could be oxidized to P+2. The second apo-protein, nifH-deletion MoFe protein, demonstrated different characteristics. The as-isolated form appears to be in the P+1 state and can be oxidized to a previously unobserved state now suggested to be S=2.0. These results indicate that nifH-deletion MoFe protein P-clusters electronically differ from the mature fully functioning P-cluster in the nifB-deletion and wild-type MoFe proteins suggesting that NifH is necessary for the maturation of the P-cluster.

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