Octahedral Iron\nin Catalytic Sites of Endonuclease\nIV from Staphylococcus aureus and Escherichia coli

During Staphylococcus aureus infections,\nreactive oxygen species cause DNA damage, including nucleotide base\nmodification. After removal of the defective base, excision repair\nrequires an endonuclease IV (Nfo), which hydrolyzes the phosphodiester\nbond 5′ to the abasic nucleotide. This class of enzymes, typified\nby the enzyme from Escherichia coli, contains a catalytic site with three metal ions, previously reported\nto be all Zn<sup>2+</sup>. The 1.05 Å structure of Nfo from the\nGram-positive organism S. aureus (<i>Sa</i>Nfo) revealed two inner Fe<sup>2+</sup> ions and one Zn<sup>2+</sup> as confirmed by dispersive anomalous difference maps. <i>Sa</i>Nfo has a previously undescribed water molecule liganded\nto Fe<sub>1</sub> forming an octahedral coordination geometry and\nhydrogen bonded to Tyr33, an active site residue conserved in many\nGram-positive bacteria, but which is Phe in Gram-negative species\nthat coordinate Zn<sup>2+</sup> at the corresponding site. The 1.9\nÅ structure of E. coli Nfo (<i>Ec</i>Nfo), purified without added metals, revealed that metal\n2 is Fe<sup>2+</sup> and not Zn<sup>2+</sup>. Octahedral coordination\nfor the sites occupied by Fe<sup>2+</sup> suggests a stereoselective\nmechanism for differentiating between Fe<sup>2+</sup> and Zn<sup>2+</sup> in this enzyme class. Kinetics and an inhibitor competition assay\nof <i>Sa</i>Nfo reveal product inhibition (or slow product\nrelease), especially at low ionic strength, caused in part by a Lys-rich\nDNA binding loop present in <i>Sa</i>Nfo and Gram-positive\nspecies but not in <i>Ec</i>Nfo. Biological significance\nof the slow product release is discussed. Catalytic activity in vitro\nis optimal at 300 mM NaCl, which is consistent with the halotolerant\nphenotype of S. aureus.