Paramagnetic intermediates of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE/IspG) under steady-state and pre-steady-state conditions.

(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE/IspG) converts 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcPP) into (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in the penultimate step of the methyl-erythritol phosphate (MEP) pathway for isoprene biosynthesis. MEcPP is a cyclic compound and the reaction involves the opening of the ring and removal of the C3 hydroxyl group consuming a total of two electrons. The enzyme contains a single [4Fe-4S] cluster in its active site. Several paramagnetic species are observed in steady-state and pre-steady-state kinetic studies. The first signal detected is from a transient species that displays a rhombic electron paramagnetic resonance (EPR) signal with g(xyz) = 2.000, 2.019, and 2.087 (FeS(A)). A second set of EPR signals (FeS(B)) accumulated during the reaction. Labeling studies with (57)Fe showed that all species observed are iron-sulfur-based. (31)P-ENDOR measurements on the FeS(A) species showed a weak (31)P coupling which is in line with binding of the substrate to the enzyme in close proximity of the active-site cluster. On the basis of the EPR/ENDOR measurements, we propose a direct binding of the substrate to the [4Fe-4S] cluster during the reaction, and therefore that the iron-sulfur cluster is directly involved in a reductive elimination of a hydroxyl group. The FeS(B) signal also showed (31)P coupling; in this case, however, it could be shown that the signal is due to the binding of the reaction product HMBPP to the active site cluster.