Structural Analysis of Protocatechuate 3,4-Dioxygenase Complexed with Product Analogs. Natesan Elango, Allen M. Orville, Michael D. Hall, John D. Lipscomb and Douglas H. Ohlendorf, Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, MN 55455, USA.

Protocatechuate 3,4-dioxygenase (3,4-PCD) utilizes mononuclear Fe(III) to catalyze the oxidative ring cleaving of 3-4-dihydroxybenzoate to produce b-carboxy-cis-cis-muconate. The Fe(III) is ligated by 2 histidines (His460, His462), 2 tyrosinates (Tyr408, Tyr447) and an exogenous solvent molecule (Wat827, thought to be an hydroxide anion) with trigonal bipyramidal geometry [1]. Previous spectroscopic and crystallographic studies [2] support a detailed reaction mechanism that includes: i) displacement of Tyr447 and Wat827 upon formation of a substrate chelate complex, ii) a conformational change which creates a sequestered dioxygen binding cavity, iii) the utilization of three exogenous ligand sites on the iron during catalysis, and iv) the reassociation of Tyr447 and Wat827 as important features of product dissociation. In the current study, crystal structures of three product complexes [glutarate, terephthalate, and fumarate] are presented. In each complex, Tyr447 is bound to iron but Wat827 is displaced. The carboxylate moiety of each product complex asymmetrically chelates the iron to yield a distorted octahedral iron coordination sphere. These results are in accord with previous spectroscopic studies and are consistent with the proposed reaction mechanism which suggests that reassociation of Tyr447 is an important step in product release.

[1] Ohlendorf, D. H., Orville, A. M. & Lipscomb, J. D. (1994) J. Mol. Biol. 244, 586-608.

[2] (a) Orville, A. M., Elango, N., Lipscomb, J. D. & Ohlendorf, D. H. (1997) submitted to Biochemistry. (b) Orville, A. M., Lipscomb, J. D. & Ohlendorf, D. H. (1997) submitted to Biochemistry.