Structural characterization of the mononuclear iron site in Pseudomonas cepacia phthalate DB01 dioxygenase using X-ray absorption spectroscopy

Abstract

Phthalate dioxygenase (PDO) from Pseudo- monas cepacia contains a Rieske-like (2Fe-2S) cluster and a mononuclear non-heme Fe(II) site. The mononu- clear iron can be replaced by a variety of divalent metal ions, although only Fe(II) permits catalytic activity. We used X-ray absorption spectroscopy to characterize the structural properties of the mononuclear 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 mea- sured directly for PDO substituted 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 evi- dence for high-Z ligands (e.g., sulfur). The relatively short average first shell bond lengths and the absence of significant outer shell scattering suggest that the mononuclear site has several oxygen ligands. With Zn in the mononuclear site, the average bond length (2.00 A) suggests a 5-coordinate site under all condi- tions. In contrast, the Co- or Fe-containing mononu- clear site appeared to be 6-coordinate and changed to 5-coordinate when substrate was bound, since the first shell bond length changed from 2.08 to 2.02 A (Co) or 2.10 to 2.06 A (Fe). The implications of these findings for the PDO mechanism are discussed.