STRUCTURAL CHARACTERIZATION OF THE MONONUCLEAR IRON SITE IN PSEUDOMONAS-CEPACIA PHTHALATE DB01 DIOXYGENASE USING X-RAY-ABSORPTION SPECTROSCOPY

Phthalate dioxygenase (PDO) from Pseudomonas cepacia contains a Rieske -like [2Fe-2S] cluster and a mononuclear non-heme Fe(II) site. The mon onuclear iron can be replaced by a variety of divalent metal ions, alt hough only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuc lear iron site and to follow the structural changes in this site as a function both of Rieske site oxidation state and of phthalate binding. Data for the mononuclear site have been measured directly for PDO sub stituted with Co or Zn in the mononuclear site, and by difference for the native 3-Fe protein. The mononuclear site was modeled well by low Z-ligation (oxygen or nitrogen) and showed no evidence for high-Z liga nds (e.g., sulfur), The relatively short average first shell bond leng ths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands, With Zn in the monon uclear site, the average bond length (2.00 Angstrom) suggests a 5-coor dinate site under all conditions. In contrast, the Co- or Fe-containin g mononuclear site appeared to be 6-coordinate and changed to 5-coordi nate when substrate was bound, since the first shell bond length chang ed from 2.08 to 2.02 Angstrom (Co) or 2.10 to 2.06 Angstrom (Fe). The implications of these findings for the PDO mechanism are discussed.