Abstract
Chemical reduction of the mononuclear ferric active site in the bacterial intradiol cleaving catecholic dioxygenase protocatechuate 3,4-dioxygenase (3,4-PCD, Brevibacterium fuscum) produces a high-spin ferrous center. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), variable-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to investigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-coordinate where the 5Eg excited-state splitting is 2033 cm-1. VTVH MCD analysis of the ground state indicates that the site has negative zero-field splitting with a small rhombic splitting of the lowest doublet (δ = 1.6 ± 0.3 cm-1). XAS pre-edge analysis also indicates a six-coordinate site while EXAFS analysis provides accurate bond lengths. Since previous spectroscopic analysis and the crystal structure of oxidized 3,4-PCD indicate a five-coordinate ferric active site, the results presented here show that the coordination number increases upon reduction. This is attributed to the coordination of a second solvent ligand. The coordination number increase relative to the oxidized site also appears to be associated with a large decrease in the ligand donor strength in the reduced enzyme due to protonation of the original hydroxide ligand.
Original language | English (US) |
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Pages (from-to) | 3676-3683 |
Number of pages | 8 |
Journal | Inorganic Chemistry |
Volume | 38 |
Issue number | 16 |
State | Published - Dec 1 1999 |