The crystal structures of protocatechuate 3,4-dioxygenase from the soil bacteria Acinetobacter sp ADP1 (Ac 3,4-PCD) has 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, with the inhibitor 4-nitrocatechol (4-NC), and with cyanide (CN) have been solved using native phases.

The overall tertiary and quaternary structure of Ac 3,4-PCD is similar to that seen in the same enzyme from Pseudomonas putida (Pp 3,4-PCD) [Ohlendorf et al. (1994) J. Mol. Biol. 244, 586-608]. At pH 8.5 the catalytic non-heme iron is coordinated by two axial ligands Y447 (147ß) and H462 (162ß) and three equatorial ligands Y408 (108ß), H460 (160ß) and a hydroxide ion (d(Fe-OH) = 1.9 Å) in a distorted bipyramidal geometry. At pH 5.75 difference maps suggest a sulfate binds to the iron in an equatorial position and the hydroxyl is shifted (d(Fe-OH = 2.3 Å) yielding octahedral geometry for the active site iron. This change in ligation geometry is concomitant with a shift in the optical absorbance spectrum of the enzyme from 450 nm (lambda max) to 520 nm (lambda max)..

Binding of substrate or 4-NC to the iron is bidentate with the axial ligand Tyr447OH (147ß) dissociating. The structure of the 4-NC complex supports the view that resonance delocalization due to the positive character of the nitrogen prevents substrate activation. The cyanide complex confirms previous work which suggested 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 catalytic function.