Abstract
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-OHINO) 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, preformed 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 site 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.
Highlights
Hyperfine broadening is observable in the EPR spec- heme iron microbial enzyme which is central to thedegradatrum of Brevibacterium fuscum protocatechuate 3,4- tive pathways for many aromatic compounds (1).Molecular dioxygenase after lyophilization and rehydration in 0’is cleaved in the catalyzedreaction and both atoms of
EPR and Mossbauer spectra show that the iron of the native enzymesis high spin ferric in an electronic environment of low symmetry (4)
Second that protocatechuate 3,4-dioxygenasesfrom B. fuscum, Pseuorder effects can result in an unsymmetrical shift in the hyperfine domonas aeruginosa, and Pseudomonas putida all react with splitting pattern giving an apparent change in the g values (18)
Summary
Substrate Complexes-The anaerobic complexes of PCA with all known protocatechuate 3,4-dioxygenasesyield EPR spectra devoid of features sufficiently sharp to detect broadening by 170-water. Inhibitor Complexes-The complex of protocatechuate 3,4&oxygenasewith 4-hydroxybenzoate, a substrate analog lackwhere D and E / D are zero field-splitting parameters, AL is the ing the 3-hydroxyl group, contains water as an iron ligand transferred hyperfine coupling tensor of the I70-ligand,and the other (Table I). Transition State Analogs-We have previously reported significant relative to the Zeeman interaction In this case, second that protocatechuate 3,4-dioxygenasesfrom B. fuscum, Pseuorder effects can result in an unsymmetrical shift in the hyperfine domonas aeruginosa, and Pseudomonas putida all react with splitting pattern giving an apparent change in the g values (18)
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