The mixed valence active center of the diiron-oxo protein uteroferrin (Ufr), a porcine purple acid phosphatase, has been analyzed by Mossbauer spectroscopy. Low and high magnetic field spectra are consistent with an antiferromagnetically spin-coupled center. Theoretical spectra that simulate experimental data have been calculated by diagonalizing the spin Hamiltonian H = JS(1) . S-2 + Sigma(i=1)(2){S-i .(g) over tilde(i) . S-i + beta S-i .(a) over tilde(i) . H + S-i .(g) over tilde(i) . I-i + I-i .(P) over tilde(i) . I-i-beta(n)g(n)H . I-i. Here the subscripts 1 and 2 refer to Fe3+ (S-1 = 5/2) and Fe2+ (S-2 = 2), respectively. For T greater than or equal to 4.2 K, both irons exhibit resolved magnetic hyperfine splittings which depend on the direction and magnitude of the external field, and the static limit of H applies. For T greater than or equal to 100 K, the spin fluctuations are fast and pure quadrupole doublets are observed unless strong fields are applied. In Ufr, the dominant isotropic exchange is strongly perturbed by the zero field splitting (ZFS). We have also calculated the EPR (g) over tilde(eff) tensor by diagonalizing the electronic terms of H. Our work confirms the claim (Sage, J. T. et al. J. Am. Chem. Sec. 1989, 111, 7239) that the anisotropy of (g) over tilde(eff) arises from the admixture of higher spin manifolds to the S-eff = 1/2 ground state by the ZFS. In order to find a solution to the Hamiltonian, we searched in its large parameter space with a genetic algorithm. This highly effective searching procedure allowed us to find an optimal parameter set that simultaneously reproduces Mossbauer spectra and EPR g values. The strategies employed for the search in the parameter space of H are discussed. We have determined the exchange constant J = 34.7 cm(-1), ZFS parameters D-1 = -0.10 cm(-1), E(1) approximate to 0, D-2 = +10.81 cm(-1), and E(2) = +3.17 cm(-1), and hyperfine tensors (a) over tilde(l)/g(n) beta(n) = -(21.4,21.2,17.8) T and (a) over tilde 2/gn beta n= -(15.2,12.2,14.1) T. We have also interpreted the 4.2 K spectra with an effective S-eff = 1/2 Hamiltonian for the ground state and determined effective hyperfine tensors (A) over tilde(1)(eff)/g(n) beta(n) = -(45.6,64.1,31.3) T and (A) over tilde(2)(eff)/g(n) beta(n) = +(11.4,24.9,16.7) T. For Fe2+, the ZFS and the electric and magnetic hyperfine interactions were consistent with the presence of axial and rhombic distortions of the dominant octahedral electrostatic potential. The principal axes of the orthorhombic field, which are rotated by 45 degrees about the z axis of the octahedral field, defined an orbital ground state for Fe2+ of x(2) - y(2)] symmetry with some z(2)] admixture, consistent with the sign of the electric field gradient. Knowledge of the orbital ground state has allowed us to estimate the intrinsic Fermi contact, orbital, and dipolar hyperfine tensors for Fe2+.