Spectroscopic Studies of the Anaerobic Enzyme−Substrate Complex of Catechol 1,2-Dioxygenase

Abstract

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme:substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UVvis) difference spectroscopic studies demonstrated that in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm −1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm −1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge-transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.

Substrate activation for O 2 reactions by oxidized metal centers in biology

“…The resting state of intradiol dioxygenases contains a highspin ferric center in a distorted trigonal bipyramidal geometry, with Tyr and His as the axial ligands and Tyr, His, and a hydroxide ligand defining the equatorial plane (20)(21)(22). Upon anaerobic substrate binding, the active site shifts to a square pyramidal geometry in which the axial Tyr and equatorial OH Ϫ are displaced by the substrate, which binds bidentate in its doubly deprotonated form (23,24). The open coordination position is trans to the equatorial His, and the substrate binds asymmetric to the Fe III center with the longer bond trans to the equatorial Tyr.…”

Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

“…Much of our mechanistic knowledge of IDOs comes from kinetic, spectroscopic, and crystallographic studies of 3,4-PCD, catechol 1,2-dioxygenase (11)(12)(13)(14)(15)(16)(17), and bio-inspired model compounds (18,19). The 3,4-PCD coordinates the Fe 3+ in a 2-His, 2-Tyr ligand set that is conserved in IDOs (11).…”

“…1 and designated 1-8 (1,13,15,17,(20)(21)(22)(23). First, PCA binds to the metal of the resting enzyme 1 in a multistep process leading to a complex 2 where both hydroxyls are ionized and the ring of PCA is axially oriented.…”

Ring-Cleaving Dioxygenases with a Cupin Fold

“…Crystal structures and X-ray absorption data revealed a ferric center in a trigonal bipyramidal geometry, with a hydroxide ligand completing the coordination sphere (31,93,94,123). The substrate binds as a dianion, donating both its protons to the displaced hydroxide and tyrosyl ligands (36,60,96). Based on spectroscopic data and electronic structure calculations, it has been proposed that the ironcatecholate interaction introduces a semiquinonate radical character to the bound substrate, which reacts directly with dioxygen to form an alkylperoxo-Fe III intermediate.…”