We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K ( M ),V (max)), EPR and (1)H, (2)H, (13)C, (31)P, and (57)Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on (57)Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a pi/sigma "metallacycle" or eta (2)-alkenyl complex. The complex is poised to interact with H(+) from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an eta (1)-allyl complex. After reduction, this forms an eta (3)-allyl pi-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by (1)H, (2)H, and (13)C ENDOR, where hyperfine couplings of approximately 6 MHz for (13)C and 10 MHz for (1)H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe(4)S(4)-containing proteins, such as IspG.