Various mechanisms have been proposed for the initial O-2 attack in intradiol dioxygenases based on different electronic descriptions of the enzyme-substrate (ES) complex. We have examined the geometric and electronic structure of the high-spin ferric ES complex of protocatechuate 3,4-dioxygenase (3,4-PCD) with UV/visible absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopies. The experimental data were coupled with DFT and INDO/S-CI calculations, and an experimentally calibrated bonding description was obtained. The broad absorption spectrum for the ES complex in the 6000-31000 cm(-1) region was resolved into at least five individual transitions, assigned as ligand-to-metal charge transfer (LMCT) from the protocatechuate (PCA) substrate and Tyr408. From our DFT calculations, all five LMCT transitions originate from the PCA and Tyr pi(op) orbitals to the ferric d pi orbitals. The strong pi covalent donor interactions dominate the bonding in the ES complex. Using hypothetical Ga3+-catecholate/semiquinone complexes as references, 3,4-PCD-PCA was found to be best described as a highly covalent Fe3+-catecholate complex. The covalency is distributed unevenly among the four PCA valence orbitals, with the strongest interaction between the pi(op-sym) and Fe d(xz) orbitals. This strong pi interaction, as reflected in the lowest energy PCA-to-Fe3+ LMCT transition, is responsible for substrate activation for the O-2 reaction of intradiol dioxygenases. This involves a multi-electron-transfer (one beta and two alpha) mechanism, with Fe3+ acting as a buffer for the spin-forbidden two-electron redox process between PCA and O-2 in the formation of the peroxy-bridged ESO2 intermediate. The Fe ligand field overcomes the spin-forbidden nature of the triplet O-2 reaction, which potentially results in an intermediate spin state (S = (3)/(2)) on the Fe3+ center which is stabilized by a change in coordination along the reaction coordinate.