Nitrogenase Cofactors

Résumé du livre

Nitrogenases provide the biochemical apparatus for the reduction of atmospheric dinitrogen (N2) to the biologically accessible form ammonia (NH3). In the case of the molybdenum nitrogenase, cleavage of the N2 triple bond occurs on the FeMoco which is synthesized outside the MoFe protein. The objective of this thesis is to elucidate several key steps in the ex situ assembly and insertion of the FeMoco into the FeMoco-deficient MoFe protein as well as the isolation and characterization the homologous FeVco from the VFe protein of the vanadium nitrogenase. The first goal of this thesis was the isolation and characterization of the FeMoco-precursor from NifEN. The NifEN-bound FeMoco-precursor represents an important mid-point in the FeMoco assembly process where the fundamental Fe-S cage has been established but the hetero-metal (Mo) and homocitrate have yet to be inserted. Isolation of the FeMoco-precursor from NifEN has allowed for the unambiguous biochemical and spectroscopic characterization of this important cofactor via activity, EPR, and XAS analysis. The second goal involved determining the role of the homocitrate in the assembly of the MoFe protein. Combined biochemical and spectroscopic analysis were used to characterize the generation of a novel iron-molybdenum-only (FeMo) cluster on NifEN which contains Mo but lacks homocitrate. The results from this investigation have helped to determine the sequence of events in the mobilization of Mo and homocitrate during FeMoco maturation as well as the role of homocitrate in the transfer of the FeMoco between NifEN and the FeMoco-deficient MoFe protein. The third goal has been to elucidate the final step of FeMoco biosynthesis where the mature FeMoco is inserted into its binding site in the MoFe protein. Point mutational analyses of a number of key residues in the MoFe protein have indicated their role in the FeMoco insertion process. The final goal of this thesis involved the extraction and characterization of the active FeVco from the alternative vanadium nitrogenase system. The biochemical and spectroscopic analysis of the isolated FeVco has allowed for the first-ever EXAFS based model as well as laying the foundation for future catalytic and structural investigations of this unique metallocluster.