Spectroscopic investigation of reduced protocatechuate 3,4-dioxygenase: Charge-induced alterations in the active site iron coordination environment

Chemical reduction of the mononuclear ferric active site in the bacterial i ntradiol 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), variab le-temperature-variable-field (VTVH) MCD, X-ray absorption (XAS) pre-edge, and extended X-ray absorption fine structure (EXAFS) spectroscopies to inve stigate the geometric and electronic structure of the reduced iron center. Excited-state ligand field CD and MCD data indicate that the site is six-co ordinate where the E-5(g) excited-state splitting is 2033 cm(-1), VTVH MCD analysis of the ground state indicates that the site has negative zero-fiel d splitting with a small rhombic splitting of the lowest doublet (delta = 1 .6 +/- 0.3 cm(-1)). XAS pre-edge analysis also indicates a six-coordinate s ite while EXAFS analysis provides accurate bond lengths. Since previous spe ctroscopic 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 de crease in the ligand donor strength in the reduced enzyme due to protonatio n of the original hydroxide ligand.