We have studied the Mössbauer spectra of reduced protocatechuate 3,4-dioxygenase (3,4-PCase) from P. aeruginosa in the temperature range from 1.5 to 200 K in applied magnetic fields up to 55 kG. The entire data set was fitted to a spin Hamiltonian pertinent to the high-spin ferrous ion. By judiciously choosing the experimental conditions, the multiparameter problem could be solved rather unambiguously. The iron sites of reduced 3,4-PCase are characterized by a negative zero-field splitting parameter D = -(6±1) cm-1 and a large rhombicity E/D = 0.25±0.05. The data show clearly that the electric field gradient tensor is rotated relative to the zero-field splitting, suggesting a point symmetry of monoclinic or lower. The electronic spin relaxation rate, at 4.2 K, is slower than 107 s-1.