Versatile peroxidase (VP) from Bjerkandera adusta, as other class II peroxidases, is inactivated by Ca2+ depletion. In this work, the spectroscopic characterizations of Ca2+-depleted VP at pH 4.5 (optimum for activity) and pH 7.5 are presented. Previous works on other ligninolytic peroxidases, such as lignin peroxidase and manganese peroxidase, have been performed at pH 7.5; nevertheless, at this pH these enzymes are inactive independently of their Ca2+ content. At pH 7.5, UV-Vis spectra indicate a heme-Fe3+ transition from 5-coordinated highspin configuration in native peroxidase to 6-coordinated low-spin state in the inactive Ca2+-depleted form. This Fe3+ hexa-coordination has been proposed as the origin of inactivation. However, our results at pH 4.5 show that Ca2+-depleted enzyme has a high spin Fe3+. EPR measurements on VP confirm the differences in the Fe3+ spin states at pH 4.5 and at 7.5 for both, native and Ca2+-depleted enzymes. In addition, EPR spectra recorded after the addition of H2O2 to Ca2+-depleted VP show the formation of compound I with the radical species delocalized on the porphyrin ring. The lack of radical delocalization on an amino acid residue exposed to solvent, W170, as determined in native enzyme at pH 4.5, explains the inability of Ca2+-depleted VP to oxidize veratryl alcohol. These observations, in addition to a notorious redox potential decrease, suggest that Ca2+-depleted versatile peroxidase is able to form the active intermediate compound I but its long range electron transfer has been disrupted.