Azotobacter vinelandii is one of a small set of diatoms possessing a nitrogenase enzyme framework capable of fixing atmospheric nitrogen to bioavailable ammonia. Electron transfer through a series of chemically novel metalloclusters with unique electrochemical properties is involved in the fixation process. The foremost of these, where substrate reduction occurs, is FeMoco, an iron-sulfur cluster containing a terminal molybdenum atom. This cluster is not generated on its target holoprotein, Av1, but instead synthesized from a iron-only precursor bound to a pre-assembly protein, nifEN, before being inserted into apo-Av1. This study investigates the incorporation of molybdenum into this iron-only precursor on nifEN and the resulting structure and activity of the matured cluster. Fits to the Mo K-Edge EXAFS spectra suggest a unique molybdenum coordination environment with greater homology to native FeMoco than previously seen, though having a distorted iron shell. EPR spectra and substrate reducing activity confirm formation of a competent cluster with similar ground state electronics as FeMoco. Finally, results implicate solution redox potential in reconstituted cluster activity and structure. The work was performed at Stanford Synchrotron Radiation Lightsource (SSRL) with support from the NIH NCRR BTP program and the US DOE BER.  SSRL operations are funded by the US DOE BES.

Molybdenum insertion into an iron-sulfur FeMoco precursor metallocluster bound on NifEN

239th National Meeting (2010)

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