Active site Fe3+ ligation by substrate and transition state analogs of protocatechuate 3,4 dioxygenase

Current proposals for the mechanism [1] of Protocatechuate (PCA) 3,4 Dioxygenase (3,4 PCD) suggest monodentate (OH) binding of PCA to the active site Fe3+. This would promote ketonization of PCA, thereby creating a carbanion at C-4 which could be directly attacked by O2. We have tested this proposal using ketonized substrate analogs and various spectroscopic probes. Our results confirm that ketonization is an essential step in the mechanism, but suggest that it occurs later in the cycle than the initial substrate complex. We have shown that water is a ligand for Brevibacterium fuscum 3,4 PCD by observing hyperfine broadening from 17OH2 on all EPR resonances of the high spin Fe3+ [2]. The spectrum of the 3,4 PCD-PCA complex is too broad to detect direct displacement of H2O by PCA. However, no broadening is obeserved in complexes with three slowly metabolized substrate analogs. In contrast, water remains bound in complexes with non-metabolized, monodentate analogs (e.g. 4-OH benzoate). Other small molecules also bind to Fe in 3,4 PCD. CN− binds in two steps; first it forms a high spin and then a low spin complex. It is likely that 2 CN− molecules bind sequentially to Fe, suggesting that there are two displaceable ligands. This is supported by the observation that PCA binds to the high spin 3,4 PCD-CN− complex to make a distinctly different ternary complex, but CN− does not bind to the, presumably bidentate, 3,4 PCD-PCA complex. The ketonized substrate analogs 2-OH-isonicotinic acid N-oxide (2-OH-INO) and 6-OH-nicotinic acid N-oxide have been synthesized [2]. These analogs form ∼100-fold stronger complexes with 3,4 PCD than does PCA, thus they are propose as transition state analogs. The EPR spectra of the 3,4 PCD-2-OH-INO complex is distinctly different than that of the CPA complex displaying small and negative zero field splitting (D = −0.5 cm−1 and intermediate rhombicity (E/D = 0.25). 17OH2 remains bound in the inhibitor complexes suggesting that they are monodentate. CN− displaces the water showing that small molecules have access to the iron in the ketonized analog complexes. Transient kinetic studies show that the ketonized analogs bind in at least two phases. In the fast initial phase, a weak, readily reversible complex is formed, while in the slow (t12 = 0.12 s) second phase, the essentially irreversible complex is formed. At −20 °C in glycerol-buffer solution two complexes can be stabilized. The first complex has optical and EPR spectral features very similar to those of the substrate complex. In contrast, the final complex is dramatically different. The native red color is bleached, due perhaps to a large blue shift of the spectrum. Similar bleached spectra are observed for early transient intermediates in the reaction with PCA [3]. We suggest that, like PCA, ketonized analogs initially assume a bidentate Fe ligation but then change to a monodentate ligation. Such a change could be coincident with a conformational change of the enzyme designed to stabilized a ketonized reaction cycle intermediate. The analogous change in the PCA complex apparently requires interactions with O2. Thus, the ketonized analogs may model the first oxy complex. Such a complex would apparently have a vacatable Fe ligand site which could be used to stabilized an oxygenous intermediate.