Large-scale quantum and molecular mechanical methods (QM/MM) and QM calculations were carried out on the soluble Delta(9) desaturase (Delta D-9) to investigate various structural models of the spectroscopically defined peroxodiferric (P) intermediate. This allowed us to formulate a consistent mechanistic picture for the initial stages of the reaction mechanism of Delta D-9, an important diferrous nonheme iron enzyme that cleaves the C-H bonds in alkane chains resulting in the highly specific insertion of double bonds. The methods (density functional theory (DFT), time-dependent DFT (TD-DFT), QM(DFT)/MM, and TD-DFT with electrostatic embedding) were benchmarked by demonstrating that the known spectroscopic effects and structural perturbation caused by substrate binding to diferrous Delta D-9 can be qualitatively reproduced. We show that structural models whose spectroscopic (absorption, circular clichroism (CD), vibrational and Miissbauer) characteristics correlate best with experimental data for the P intermediate correspond to the mu-1,2-O-2(2-) binding mode. Coordination of Glu196 to one of the iron centers (Fe-B) is demonstrated to be flexible, with the monodentate binding providing better agreement with spectroscopic data, and the bidentate structure being slightly favored energetically (1-10 kJ mol(-1)). Further possible structures, containing an additional proton or water molecule are also evaluated in connection with the possible activation of the P intermediate. Specifically, we suggest that protonation of the peroxide moiety, possibly preceded by water binding in the Fe-A coordination sphere, could be responsible for the conversion of the P intermediate in Delta D-9 into a form capable of hydrogen abstraction. Finally, results are compared with recent findings on the related ribonudeotide reductase and toluene/methane monooxygenase enzymes.