Crystal structures of substrate and substrate analog complexes of protocatechuate 3,4-dioxygenase: Endogenous Fe³⁺ ligand displacement in response to substrate binding

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 β-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 Å 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 (147β). After its release from the iron, Tyr447 is stabilized by hydrogen bonding to Tyr16 (16α) and Asp413 (113β) 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·NO 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., and 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.