Dimerization of the global anaerobic transcription factor FNR is essential for FNR activity. Under aerobic conditions FNR is an inactive monomeric species because it lacks the oxygen labile [4Fe-4S] cluster required for dimerization. In this study, we investigated the protein side chains that inhibit FNR dimerization under aerobic conditions. Substitution of Asp(154) within the predicted dimerization helix with residues containing neutral or positively charged side chains increased FNR activity under aerobic conditions, whereas replacement of Asp(154) with Glu inhibited FNR activity similar to WT-FNR. Similar results were obtained when making analogous substitutions of Glu(150). In vitro analysis of representative FNR mutant proteins indicated that their increased activity under aerobic conditions resulted from an [4Fe-4S] independent mechanism of dimerization. In addition, simultaneous substitution of residues 150 and 154 with Lys restored inhibition of FNR activity under aerobic growth conditions. Collectively, these data indicate that charge repulsion by side chains at positions 150 and 154 is necessary to inhibit dimerization under aerobic conditions. They also suggest that a [4Fe-4S]-dependent conformational change overcomes charge repulsion between subunits under anaerobic conditions. Comparison of the trypsin sensitivity of [4Fe-4S]-FNR and apoFNR indicated that there are no major differences in protease sensitivity between these forms, whereas circular dichroism suggested that small changes in secondary structure occur between the cluster-containing FNR and apoFNR. Thus, the [4Fe-4S]-dependent conformational change necessary to overcome inter-subunit charge repulsion and create a subunit interface more favorable for dimerization must be small.