The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K-i = 20 +/- 2, nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K-i = 54 +/- 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex.