Simultaneous binding of nitric oxide and isotopically labeled substrates or inhibitors by reduced protocatechuate 3,4-dioxygenase.
The active site Fe3+ of protocatechuate (PCA) 3,4-dioxygenase can be nonenzymatically reduced to Fe2+, to give a colorless and EPR-silent enzyme (Er). Nitric oxide (NO) binds to Er to yield a species with EPR (S = 3/2; g = 4.341, 3.693, 1.984; E/D = 0.055) and optical absorption (lambda max = 430 nm, epsilon approximately 1870 M-1 cm-1/iron) spectra. Addition of NO to a preformed Er. PCA complex results in a new species (EPR: S = 3/2; g = 4.920, 2.988, 1.846; E/D = 0.175; optical: lambda max = 404 nm, epsilon approximately 3930 M-1 cm-1/iron). Hyperfine broadening from the substrates [17O]PCA or [17O]homoprotocatechuate (HPCA) is observed in the EPR spectra of Er.substrate.NO complexes only when the 17O (I = 5/2) is placed in the carbon-4 OH group, suggesting that only this group binds to the iron when NO is bound. Previous studies (Orville, A. M., and Lipscomb, J. D. (1989) J. Biol. Chem. 261, 8791-8801) showed that both OH groups of HPCA can bind to the Fe3+ of the oxidized enzyme. Thus, the NO may compete with the substrate carbon-3 OH group for a binding site on the Fe2+. In contrast, when either PCA or HPCA is added to a preformed Er.NO complex, no substrate binding to the Fe2+ is detected. At 2.3 K, white light photodissociates NO from the Er.NO and Er.PCA.NO complexes. The Er.NO complex is photodissociated to a greater extent than the Er.PCA.NO complex, and different NO rebinding kinetics are observed showing that the substrate strongly influences the photodissociation/reassociation process. Photodissociation of each complex results in the formation of some Fe3+, suggesting that the nitrosyl complex has at least partial Fe(3+)-NO- character. In solution at 5-10 degrees C, white light promotes conversion of preformed Er.NO plus PCA to the Er.PCA.NO complex, suggesting that formation of the latter complex requires dissociation of NO. It is proposed that initial NO binding blocks the single site for exogenous ligand binding on the iron, thereby inhibiting PCA association. In contrast, PCA binding before NO appears to evoke an enzyme conformational change that allows simultaneous NO binding in another ligand site. These results are consistent with the current model for the mechanism of intradiol dioxygenases in which a PCA-induced conformational change allows substrate to bind as an Fe3+ chelate and O2 reacts initially with the PCA rather than the Fe3+.
Department of Biochemistry, Medical School, University of Minnesota, Minneapolis 55455.
A M
AM
Orville
J D
JD
Lipscomb
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D062385:Hydroxybenzoates / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007539:Isonicotinic Acids / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D011520:Protocatechuate-3,4-Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D062385:Hydroxybenzoates; D007539:Isonicotinic Acids; D009569:Nitric Oxide; C009091:protocatechuic acid; D007501:Iron; D011520:Protocatechuate-3,4-Dioxygenase