The dinitrogen-binding site in the Mo-based nitrogenase is FeMo-cofactor, a metallo-sulfur cluster of composition MoFe7S9. R-homocitrate. The NifV(-) mutant nitrogenase from Klebsiella pneumoniae contains an FeMo-cofactor in which homocitrate has been replaced by citrate (i.e., MoFe7S9. citrate). Both the wild type and mutant cofactors (in the S = 3/2 spin state) can be extracted into N-methylformamide. The extracted cofactors bind one molecule of PhS- at the tetrahedral Fe, and the rate of this reaction depends on what else is coordinated to the cluster. No differences were observed between the reactivities of wild-type and NifV(-) cofactors with PhS- when they were complexed with CN-, N-3(-), or H+ However, when imidazole is bound, the kinetics of the reactions of PhS- with the two cofactors are very different. Here we propose that R-homocitrate (but not citrate) can hydrogen bond to the imidazole ligand on Mo, and that this perturbs the electron distribution within the cluster core, and hence its reactivity with PhS-. Using the X-ray crystallographic data for the MoFe-protein of nitrogenase and molecular mechanics calculations, we have investigated the implications of these findings on the action of the enzyme. Our model shows that R-homocitrate is uniquely capable of facilitating the binding of dinitrogen by allowing the substrate access to Mo after dissociation of the Mo-carboxylate bond while simultaneously influencing the electron-richness of the cofactor by hydrogen bonding of the pendant -CH2CH2CO2 arm to the imidazole group of His alpha 442. The whole process is mediated by hydrogen bonding of amino acid side chains to the carboxylate groups of R-homocitrate.