Magnetic circular dichroism studies of exogenous ligand and substrate binding to the non-heme ferrous active site in phthalate dioxygenase

• Published in: Chemistry & biology Vol. 1; no. 3; pp. 173 - 183
• Main Authors: Pavel, Elizabeth G., Martins, Laura J., Ellis, Walther R., Solomon, Edward I.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Ltd 01.11.1994
• Subjects: Binding Sites; Circular Dichroism; Ferrous Compounds - chemistry; Ferrous Compounds - metabolism; Ligands; NIR MCD; non-heme ferrous; Oxygenases - chemistry; Oxygenases - metabolism; phthalate dioxygenase; Rieske; Substrate Specificity; phthalate dioxygenase; Rieske; non-heme ferrous; NIR MCD
• ISSN: 1074-5521; 1879-1301
• DOI: 10.1016/1074-5521(94)90007-8

Background: Mononuclear non-heme iron centers are found in the active sites of a variety of enzymes that require molecular oxygen for catalysis. The mononuclear non-heme iron is believed to be the active site for catalysis, and is presumed to bind and activate molecular oxygen. The mechanism of this reaction is not understood. Phthalate dioxygenase is one such enzyme. Because it also contains a second iron site, the Rieske site, it is difficult to obtain information on the structure of the active site. We therefore used magnetic circular dichroism (MCD) spectroscopy to probe the mononuclear non-heme Fe 2+ site in this biodegradative enzyme. Results: The MCD spectrum of the resting enzyme shows features indicative of one six-coordinate Fe 2+ site; substrate binding converts the site to two different five-coordinate species, opening up a coordination position for O 2 binding. MCD spectra of the corresponding apoenzyme have been subtracted to account for temperature-independent contributions from the Rieske site. Azide binds both to the resting enzyme to produce a new six-coordinate species, showing that one of the ferrous ligands is exchangeable, and also to the enzyme-substrate complex to form a ternary species. The low azide binding constant for the substrate -enzyme species relative to the resting enzyme indicates steric interaction and close proximity between exogerious ligand and the substrate. Conclusions: We have been able to provide some detailed structural insight into exogenous ligand and substrate binding to the non-heme Fe 2+ site, even in the presence of the enzyme's [2Fe2S] Rieske center. Further mechanistic studies are now required to maximize the molecular-level detail available from these spectroscopic studies.