Weixue Wang - Bioorganometallic mechanism of action, and inhibition, of IspH

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      Publication Details (including relevant citation   information):

      PNAS  2010 107 (10)  4522-4527; published   ahead of print February 19, 2010,  doi:10.1073/pnas.0911087107


      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   1H, 2H, 13C, 31P, and   57Fe-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 57Fe   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   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 diphosphates (such as   propargyl diphosphate) are potent IspH inhibitors and likewise   form metallacycle complexes, as evidenced by 1H,   2H, and 13C ENDOR, where hyperfine   couplings of approximately 6 MHz for 13C 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   Fe4S4-containing proteins, such as IspG.

      Address (URL): http://www.pnas.org/content/107/10/4522.full