Structure of Acinetobacter Strain ADP1 Protocatechuate 3,4-Dioxygenase at 2.2 Å Resolution: Implications for the Mechanism of an Intradiol Dioxygenase

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Abstract
The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacter strain ADP1 (Ac 3,4-PCD) have been determined in space group I23 at pH 8.5 and 5.75. In addition, the structures of Ac 3,4-PCD complexed with its substrate 3,4-dihydroxybenzoic acid (PCA), the inhibitor 4-nitrocatechol (4-NC), or cyanide (CN-) have been solved using native phases. The overall tertiary and quaternary structures of Ac 3,4-PCD are similar to those of the same enzyme from Pseudomonas putida [Ohlendorf et al. (1994) J. Mol. Biol. 244, 586−608]. At pH 8.5, the catalytic non-heme Fe3+ is coordinated by two axial ligands, Tyr447OH (147β) and His460Nε2 (160β), and three equatorial ligands, Tyr408OH (108β), His462Nε2 (162β), and a hydroxide ion (d(Fe−OH) = 1.91 Å) in a distorted bipyramidal geometry. At pH 5.75, difference maps suggest a sulfate binds to the Fe3+ in an equatorial position and the hydroxide is shifted [d(Fe−OH) = 2.3 Å] yielding octahedral geometry for the active site Fe3+. This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from λmax = 450 nm to λmax = 520 nm. Binding of substrate or 4-NC to the Fe3+ is bidentate with the axial ligand Tyr447OH (147β) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization of the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work that protocatechuate 3,4-dioxygenases have three coordination sites available for binding by exogenous substrates. A significant conformational change extending away from the active site is seen in all structures when compared to the native enzyme at pH 8.5. This conformational change is discussed in its relevance to enhancing catalysis in protocatechuate 3,4-dioxygenases.