The electronic structure of Fe3+ was studied in a mutant that has been modified to bind manganese or iron at a site corresponding to the manganese-binding site of photosystem II (Kalman et al., Biochemistry 45:13869–13874, 2006). Using electron paramagnetic resonance spectroscopy, the presence of the oxidized state of the bacteriochlorophyll dimer, P·+, was detected in the light when no metal was added. When iron was bound to the modified reaction centers in the presence of bicarbonate, the contribution of P·+ was greatly reduced and a signal characteristic of Fe3+ was evident. To characterize the electronic structure of this ferric ion, the electron paramagnetic resonance spectrum was measured at X-band at temperatures from 4 to 200 K. The major contribution to the spectrum at 4 K is from Fe3+ with a spin 3/2 in a rhombic coordination and E/D ratio of 0.1914 and g eff values of 6.0, 2.9, and 2.0. As the temperature increases from 4 to 200 K, the signal shifts, with the central g eff value changing from 2.9 to 2.2. This change with temperature may result from alterations in the interaction with the bicarbonate coordinated to the iron as the temperature increases.