Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases.
Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D001665:Binding Sites; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D062385:Hydroxybenzoates / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010226:Parabens; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology
D001426:Bacterial Proteins; D062385:Hydroxybenzoates; D010226:Parabens; C032948:3-hydroxybenzoic acid; C009091:protocatechuic acid; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; C022476:protocatechuate 4,5-dioxygenase; C038193:4-hydroxybenzoic acid; D010100:Oxygen