Crystal Structures of Substrate and Substrate Analog Complexes of Protocatechuate 3,4-Dioxygenase:  Endogenous Fe³⁺ Ligand Displacement in Response to Substrate Binding^,

Abstract

Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes a ferric ion to catalyze the aromatic ring cleavage of 3,4-dihydroxybenzoate (PCA) by incorporation of both atoms of dioxygen to yield beta-carboxy-cis, cis-muconate. The crystal structures of the anaerobic 3,4-PCD.PCA complex, aerobic complexes with two heterocyclic PCA analogs, 2-hydroxyisonicotinic acid N-oxide (INO) and 6-hydroxynicotinic acid N-oxide (NNO), and ternary complexes of 3,4-PCD.INO.CN and 3,4-PCD. NNO.CN have been determined at 2.1-2.2 A resolution and refined to R-factors between 0.165 and 0.184. PCA, INO, and NNO form very similar, asymmetrically chelated complexes with the active site Fe3+ that result in dissociation of the endogenous axial tyrosinate Fe3+ ligand, Tyr447 (147beta). After its release from the iron, Tyr447 is stabilized by hydrogen bonding to Tyr16 (16alpha) and Asp413 (113beta) and forms the top of a small cavity adjacent to the C3-C4 bond of PCA. The equatorial Fe3+ coordination site within this cavity is unoccupied in the anaerobic 3,4-PCD.PCA complex but coordinates a solvent molecule in the 3,4-PCD.INO and 3,4-PCD.NNO complexes and CN- in the 3,4-PCD.INO.CN and 3,4-PCD.NNO.CN complexes. This shows that an O2 analog can occupy the cavity and suggests that electrophilic O2 attack on PCA is initiated from this site. Both the dissociation of the endogenous Tyr447 and the expansion of the iron coordination sphere are novel features of the 3,4-PCD. substrate complex which appear to play essential roles in the activation of substrate for O2 attack. Together, the structures presented here and in the preceding paper [Orville, A. M., Elango, N. , Lipscomb, J. D., & Ohlendorf, D. H. (1997) Biochemistry 36, 10039-10051] provide atomic models for several steps in the reaction cycle of 3,4-PCD and related Fe3+-containing dioxygenases.

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.…”

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

“…Nevertheless, the negative 1600 cm −1 feature of the difference spectrum obtained with HPNO indicates that the binding of this compound induces tyrosinate protonation, consistent with previous demonstrations that such N-oxide inhibitors cause tyrosinate dissociation in PCD. 20 The UVRR spectrum of HPNO − (Figure 3e) differs from that of catechol ( Figure 3a-d) in that it contains strong features around 1180 cm −1 . It is likely that these features mask the signal of the expected negative tyrosinate bands in this region of the HPNO difference spectrum.…”

“…17 , 20 An interaction between a highly conserved arginine residue and the catecholate oxygen of the long Fe-O bond may also be involved in inducing asymmetry. 20 The importance of this arginine residue has been demonstrated by the inability to grow on 4-hydroxybenzoate of an Acinetobacter strain containing an Arg457Ser mutant of protocatechuate 3,4-dioxygenase (PCD).30 Finally, it is possible that the asymmetric binding reflects ketonization of the substrate.…”

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

“…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.…”