Cyanide and nitric oxide binding to reduced protocatechuate 3,4- dioxygenase: Insight into the basis for order-dependent ligand binding by intradiol catecholic dioxygenases

EPR-silent, chemically reduced protocatechuate 3,4-dioxygenase (E(r)) binds NO at the active site Fe²⁺ to yield an EPR-active, S = 3/2 species that blocks subsequent binding of all other exogenous ligands. In contrast, addition of NO to a preformed E(r)·CN^- complex yields an EPR-active, S = 1/4 species [E(r)·(CN)(x)·NO] that exhibits resolved superhyperfine splitting from ¹³CN^-, 15/14NO, and a protein-derived ¹⁴N. Simulations of the EPR spectra observed for the E(r)·(CN)(x)·NO complex formed with ¹²CN^- and ¹³CN^- (1:1) show that CN^- binds in two iron ligand sites (x ≤ 2). The two cyanides exhibit similar, but distinguishable, hyperfine coupling constants. This demonstrates unambiguously that at least three exogenous ligands (two cyanides and NO) can bind to the Fe²⁺ simultaneously and strongly suggests that at least one histidine ligand is retained in the complex. The E(r)·(CN)(≤2)·NO complex readily exchanges both of the bound cyanides for the substrate analog, 2-hydroxyisonicotinic acid N-oxide (INO), to form a E(r)·INO·NO complex exhibiting the same S = 3/2 type EPR spectrum that is observed for this complex in the absence of CN^-. Because the dead- end E(r)·NO complex does not accumulate during the exchange, the results suggest that E(r)·(CN)(≤2)·NO and E(r)·INO·NO are in conformational states that allow facile exchange of INO and CN^- but not NO. The results are interpreted in the context of the known X-ray crystal structures for the ferric form of the resting enzyme (E(ox)) and numerous E(ox)·substrate, inhibitor, and CN^- complexes, all of which have a charge neutral iron center. It is proposed that the binding of one CN^- causes dissociation of an anionic endogenous ligand which begins a series of conformational changes analogous to those initiated by anionic substrate binding to E(ox). This results in a new unique coordination site for NO, and a new second site for CN^-; both cyanide sites are utilized when the enzyme subsequently binds substrates or INO.