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 (Er) 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 Er.CN- complex yields an EPR-active, S = 1/2 species [Er.(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 Er.(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 Er.(CN)>/=2.NO complex readily exchanges both of the bound cyanides for the substrate analog, 2-hydroxyisonicotinic acid N-oxide (INO), to form a Er.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 Er.NO complex does not accumulate during the exchange, the results suggest that Er.(CN)>/=2. NO and Er.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 (Eox) and numerous Eox.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 Eox. 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.
Orville
A M
AM
Department of Biochemistry, Medical School, and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455-0347, USA.
Lipscomb
J D
JD
D003486:Cyanides / Q000737:chemistry / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron; D008024:Ligands; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011520:Protocatechuate-3,4-Dioxygenase / Q000737:chemistry / Q000378:metabolism*
D003486:Cyanides; D008024:Ligands; D009569:Nitric Oxide; D007501:Iron; D011520:Protocatechuate-3,4-Dioxygenase