Superoxide dismutases (SODs) are proteins specialized in the depletion of superoxide from the cell through disproportionation of this anion into oxygen and hydrogen peroxide. We have used high-field electron paramagnetic resonance (HFEPR) to test a two-site binding model for the interaction of manganese-SODs with small anions. Because tyrosine-34 was thought to act as a gate between these two sites in this model, a tyrosine to phenylalanine mutant of the superoxide dismutase from R. capsulatus was constructed. Although the replacement slightly reduced activity, HFEPR measurements demonstrated that the electronic structure of the Mn(II) center was unaffected by the mutation. In contrast, the mutation had a profound effect on the interactions of fluoride and azide with the Mn(II) center. It was concluded that the absence of tyrosine-34 prevented the close approach of these anions to the metal ion. This mutation also enhanced the formation of a hexacoordinated water-Mn(II)SOD complex at low temperatures. Together, these results showed that the role of Y34 is unlikely to involve redox tuning but rather is important in regulating the equilibria between the anionic substrate in solution and the two binding sites near the metal. These observations further supported the originally proposed mutually exclusive two-binding-site model.