¹⁷O-water and cyanide ligation by the active site iron of protocatechuate 3,4-dioxygenase. Evidence for displaceable ligands in the native enzyme and in complexes with inhibitors or transition state analog

Hyperfine broadening is observable in the EPR spectrum of Brevibacterium fuscum protocatechuate 3,4-dioxygenase after lyophilization and rehydration in ¹⁷O-enriched water, demonstrating H₂O ligation to the active site iron. Lack of detectable broadening in the sharp features of the spectra of three substrate complexes suggests that H₂O is displaced by substrate. Water is bound in the monodentate complex with the competitive inhibitor 3-hydroxybenzoate which binds directly to the iron showing that two iron ligation sites can be occupied by nonprotein ligands. Ketonized substrate analogs which mimic a proposed transition state of the reaction cycle, 2-hydroxyisonicotinic acid N-oxide (2-OH INO) and 6-hydroxynicotinic acid N-oxide (6-OH NNO), have H₂O bound in their final, bleached enzyme complexes, suggesting that these complexes are also monodentate. In contrast, a transient, initial complex of 6-OH NNO which is spectrally similar to the substrate complex, apparently does not have H₂O bound. Cyanide binding occurs in two steps. The active site Fe³⁺ of the initial, rapidly formed, violet complex is high spin while that of the second, slowly formed, green complex is low spin; a unique state for mononuclear non-heme iron enzymes. The data suggest that the Fe-CN^- and Fe-(CN^-)₂ complexes form sequentially. CN^- binds to enzyme complexes with 2-OH INO and 6-OH NNO in one step to yield high spin Fe³⁺ species. In contrast, performed substrate complexes prevent Cn^- binding. CN^- binding eliminates the broadening due to ¹⁷O-water in the EPR spectra of both native enzyme and the enzyme-ketonized analog complexes. A model is proposed in which H₂O is displaced by bidentate binding of the substrate but can potentially rebind after a subsequent substrate ketonization. The proximity of the vacatable H₂O-binding sites of the iron to the site of oxygen insertion suggests, however, that this site may serve to stabilize an oxygenated intermediate during the reaction cycle.