Bioorganometallic mechanism of action, and inhibition, of IspH

摘要

We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) re-ductase], together with its inhibition, using a combination of site-directed mutagenesis (K_M, V_(max)). EPR and ~1H, ~2H, ~(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, ethy-lene .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 organo-metallic species with HMBPP, a π/σ "metallacycle" or η~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 η~1 -allyl complex. After reduction, this forms an η~3-allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne dipho-sphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by ~1H, ~2H, and ~(13)C ENDOR, where hyperfine couplings of approximately 6 MHz for ~(13)C and 10 MHz for ~1H, 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_4S_4-containing proteins, such as IspG.