Biological nitrogen fixation is catalyzed by nitrogenase, an enzyme composed of two component proteins called the Fe protein and the MoFe protein. During catalysis, electrons are delivered one at a time from the Fe protein to the MoFe protein in a process involving component-protein association and dissociation and hydrolysis of at least two MgATP for each electron transfer. The Fe protein contains the sites for MgATP binding and hydrolysis, whereas the site for substrate binding and reduction is located on the MoFe protein. Among the important aspects of nitrogenase enzymology discussed here are (a) the structures of the metal centers that participate in electron transfer, (b) the organization of the metalloclusters within the polypeptides and their contributions to substrate binding and electron transfer, (c) the nature of the dynamic interactions between the two component proteins that lead to nucleotide hydrolysis and intermolecular electron transfer, (d) the mechanism by which the multiple electrons necessary for substrate reduction are distributed within the MoFe protein, (e) the nature of the intramolecular electron path within the MoFe protein, and (f) where and how substrate and various inhibitors become bound to the substrate-reduction site. This chapter summarizes biochemical-genetic strategies used to address these questions and discussed them in the context of the recently proposed three-dimensional models for both the Fe protein and MoFe protein from Azotobacter vinelandii.