Octahedral Iron in Catalytic Sites of Endonuclease IV from <i>Staphylococcus aureus</i> and <i>Escherichia coli</i>

During <i>Staphylococcus aureus</i> infections, reactive oxygen species cause DNA damage, including nucleotide base modification. After removal of the defective base, excision repair requires an endonuclease IV (Nfo), which hydrolyzes the phosphodiester bond 5' to the abasic nucleotide. This class of enzymes, typified by the enzyme from <i>Escherichia coli</i>, contains a catalytic site with three metal ions, previously reported to be all Zn²⁺. The 1.05 Å structure of Nfo from the Gram-positive organism <i>S. aureus</i> (<i>Sa</i>Nfo) revealed two inner Fe²⁺ ions and one Zn²⁺ as confirmed by dispersive anomalous difference maps. <i>Sa</i>Nfo has a previously undescribed water molecule liganded to Fe₁ forming an octahedral coordination geometry and hydrogen bonded to Tyr33, an active site residue conserved in many Gram-positive bacteria, but which is Phe in Gram-negative species that coordinate Zn²⁺ at the corresponding site. The 1.9 Å structure of <i>E. coli</i> Nfo (<i>Ec</i>Nfo), purified without added metals, revealed that metal 2 is Fe²⁺ and not Zn²⁺. Octahedral coordination for the sites occupied by Fe²⁺ suggests a stereoselective mechanism for differentiating between Fe²⁺ and Zn²⁺ in this enzyme class. Kinetics and an inhibitor competition assay of <i>Sa</i>Nfo reveal product inhibition (or slow product release), especially at low ionic strength, caused in part by a Lys-rich DNA binding loop present in <i>Sa</i>Nfo and Gram-positive species but not in <i>Ec</i>Nfo. Biological significance of the slow product release is discussed. Catalytic activity in vitro is optimal at 300 mM NaCl, which is consistent with the halotolerant phenotype of <i>S. aureus</i>.