[17O]Water and nitric oxide binding by protocatechuate 4,5-dioxygenase and catechol 2,3-dioxygenase. Evidence for binding of exogenous ligands to the active site Fe2+ of extradiol dioxygenases.
Pseudomonas testosteroni protocatechuate 4,5-dioxygenase and Pseudomonas putida catechol 2,3-dioxygenase (metapyrocatechase) catalyze extradiol-type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme 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 complexes by 17O-enriched H2O shows that water is bound directly to the Fe2+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by 17O-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex. Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KD values of 190 microM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a KD for NO of 3.4 microM. One type of NO binding site is observed for both the native and substrate complexed protocatechuate 4,5-dioxygenase with KD values of 360 and 3 microM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.
D001665:Binding Sites; D050557:Catechol 2,3-Dioxygenase; D049308:Dioxygenases; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010103:Oxygen Isotopes; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D014867:Water / Q000378:metabolism*
D010103:Oxygen Isotopes; D014867:Water; D009569:Nitric Oxide; D007501:Iron; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase; C022476:protocatechuate 4,5-dioxygenase