Variable-Temperature Variable-Field Magnetic Circular Dichroism Studies of the Fe(II) Active Site in Metapyrocatechase: Implications for the Molecular Mechanism of Extradiol Dioxygenases

Patricia A. Mabrouk, Allen M. Orville, John D. Lipscomb, Edward I. Solomon

Research output: Contribution to journalArticle

77 Scopus citations

Abstract

A combination of optical absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectroscopies has been used to probe the geometric and electronic structure of the catalytically relevant Fe2+ active site in metapyrocatechase (catechol 2,3-dioxygenase). The number and energies of the excited-state ligand field features have been used to define the effective active site geometry. The magnetic field and temperature dependence of the MCD intensity of the excited-state ligand field features have been used to determine the ground-state zero-field splitting and g values for the EPR inactive S = 2 center. Ligand field diagrams have been constructed on the basis of experimental estimates of the e and t2 d orbital splittings derived from the excited-state and ground-state analyses, respectively. A 5-coordinate square-pyramidal effective geometry is obtained for the active site of resting metapyrocatechase. Spectroscopic evidence is presented for the binding of substrate (catechol) to the active site Fe2+. Catechol appears to bind in a bidentate fashion, occupying the axial site and one equatorial position at the ferrous center. Addition of azide to the enzyme causes no change in the MCD spectrum, suggesting that azide does not bind to the iron. However, substrate binding to the iron activates the enzyme toward azide binding. A square-pyramidal geometry is also found for this ternary complex, with azide occupying an equatorial ligation site at the ferrous center. Geometric and electronic structural changes resulting from the addition of substrate and azide to resting metapyrocatechase are discussed in terms of their relevance to the catalytic mechanism for the extradiol dioxygenases.

Original languageEnglish (US)
Pages (from-to)4053-4061
Number of pages9
JournalJournal of the American Chemical Society
Volume113
Issue number11
DOIs
StatePublished - May 1 1991

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