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 ((E(r)). Nitric oxide (NO) binds to E(r) to yield a species with EPR (S = 3/2; g = 4.341, 3.693, 1.984; E/D = 0.055) and optical absorption (λ(max) = 430 nm, ε ≃ 1870 M^-1 cm^-1/iron) spectra. Addition of NO to a preformed E(r) · PCA complex results in a new species (EPR: S = 3/2; g = 4.920, 2.988, 1.846; E/D = 0.175; optical: λ(max) = 404 nm, ε ≃ 3930 M^-1 cm^{- 1}/iron). Hyperfine broadening from the substrates [¹⁷O]PCA or [¹⁷O]homoprotocatechuate (HPCA) is observed in the EPR spectra of E(r) · 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 E(r) · NO complex, no substrate binding to the Fe²⁺ is detected. At 2.3 K, white light photodissociates NO from the E(r) · NO and E(r) · PCA · NO complexes. The E(r) · NO complex is photodissociated to a greater extent than the E(r) · 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 Fe³⁺- NO^- character. In solution at 5-10 °C, white light promotes conversion of preformed E(r) · NO plus PCA to the E(r) · 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⁺.