Nitrogenase is the only enzyme that can convert atmospheric dinitrogen (N-2) into biologically usable ammonia (NH3). To achieve this multielectron redox process, the nitrogenase component proteins, MoFe-protein (MoFeP) and Fe-protein (FeP), repeatedly associate and dissociate in an ATP-dependent manner, where one electron is transferred from FeP to MoFeP per association. Here, we provide experimental evidence that encounter complexes between Fe? and MoFeP play a functional role in nitrogenase catalysis. The encounter complexes are stabilized by electrostatic interactions involving a positively charged patch on the beta-subunit of MoFeP. Three single mutations (beta Asn399Glu, beta Lys400Glu, and beta Arg401Glu) in this patch were generated in Azotobacter vinelandii MoFeP. All of the resulting variants displayed decreases in specific catalytic activity, with the beta K400E mutation showing the largest effect. As simulated by the Thomeley-Lowe kinetic scheme, this single mutation lowered the rate constant for FeP-MoFeP association S-fold. We also found that the beta K400E mutation did not affect the coupling of ATP hydrolysis with electron transfer (ET) between FeP and MoFeP. These data suggest a mechanism where FeP initially forms encounter complexes on the MoFeP beta-subunit surface en route to the ATP-activated, ET-competent complex over the alpha beta-interface.