MAGNETIC CIRCULAR-DICHROISM STUDIES ON THE MONONUCLEAR FERROUS ACTIVE-SITE OF PHTHALATE DIOXYGENASE FROM PSEUDOMONAS-CEPACIA SHOW A CHANGE OF LIGATION STATE ON SUBSTRATE-BINDING

Phthalate dioxygenase from Pseudomonas cepacia contains a mononuclear ferrous center that is strictly required for catalytic oxygen activati on. The spectroscopic characterization of this iron site and its ligan d interactions has been complicated in the past by interference from a Rieske-type binuclear (2Fe-2S) cluster in the enzyme, which dominates the absorption spectra and is superimposed in X-ray absorption spectr a for the mononuclear site. We have used low-temperature, variable mag netic field circular dichroism spectroscopy to selectively detect the ligand field spectra of the paramagnetic mononuclear ferrous active si te in the presence of the diamagnetic exchange-coupled Rieske center a nd observe spectral changes associated with substrate binding, The per turbations of the d --> d spectra for the mononuclear ferrous site ref lect a decrease in coordination number from six to five on binding sub strate. This structural change suggests that displacement of an iron l igand prepares the ferrous center for dioxygen activation.