Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*<or= 0.23 nM (EI*). In sharp contrast, the previously reported small molecule PDF inhibitor, SB-543668, is a competitive, readily reversible inhibitor (t(1/2) for dissociation = 2.8 s). In addition, we demonstrate that BB-3497 is also a time-dependent inhibitor of PDF with an extremely slow off-rate. The two-step inhibition model detailed herein for the inhibition of Staphylococcus aureus PDF by actinonin and BB-3497 is consistent with a recent report on the time-dependent inhibition of Escherichia coli PDF by a macrocyclic peptidomimetic inhibitor [Ngugen, K. T., et al. (2004) Bioorg. Chem. 32, 178-191]. This study substantially extends our understanding of PDF inhibition and may facilitate the development of novel antibiotics.