Strategies for the Functional Analysis of the Azotobacter Vinelandii MoFe Protein and its Active Site FeMo-Cofactor

The nitrogenase MoFe protein is the key player in Mo-dependent biological nitrogen fixation because it provides the site of substrate binding and reduction (Burgess and Lowe, 1996). As described elsewhere in this volume, reducing equivalents are delivered during catalysis from the Fe protein to the MoFe protein in a reaction coupled to MgATP hydrolysis. Although the nucleotide-binding sites are located on the Fe protein, it does not catalyze MgATP hydrolysis in the absence of the MoFe protein. Also, neither the Fe protein nor the MoFe protein exhibits any substrate-reduction activity in the absence of its catalytic partner. An overall view of the role of MgATP in nitrogenase catalysis suggests a dynamic process involving transmission of signals back and forth from the MgATP site to the docking surface of the Fe protein, and probably extends into the MoFe protein and back again from the MoFe protein into the Fe protein (Howard and Rees, 1994). It seems likely that this complicated series of events ensures the unidirectional flow of electrons from the Fe protein to their final destination of substrates. Electrons delivered from the Fe protein appear to be initially received by an unusual [8Fe-7S] metallocluster, called the P cluster and these electrons are then delivered to another novel metallocluster, called the FeMo-cofactor [7Fe-9S-Mo-X-homocitrate], which provides the substrate-binding and -reduction site.