UC San Diego

The recently discovered methylerythritol phosphate (MEP) pathway provides new targets for the development of antibacterial and antimalarial drugs. In the final step of the MEP pathway, the [4Fe-4S] IspH protein catalyzes the 2e(-)/2H(+) reductive dehydroxylation of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) to afford the isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). Recent experiments have attempted to elucidate the IspH catalytic mechanism to drive inhibitor development. Two competing mechanisms have recently emerged, differentiated by their proposed HMBPP binding modes upon 1e(-) reduction of the [4Fe-4S] cluster: (1) a Birch reduction mechanism, in which HMBPP remains bound to the [4Fe-4S] cluster through its terminal C4-OH group (ROH-bound) until the -OH is cleaved as water; and (2) an organometallic mechanism, in which the C4-OH group rotates away from the [4Fe-4S] cluster, allowing the HMBPP olefin group to form a metallacycle complex with the apical iron (η(2)-bound). We perform broken-symmetry density functional theory computations to assess the energies and reduction potentials associated with the ROH- and η(2)-bound states implicated by these competing mechanisms. Reduction potentials obtained for ROH-bound states are more negative (-1.4 to -1.0 V) than what is typically expected of [4Fe-4S] ferredoxin proteins. Instead, we find that η(2)-bound states are lower in energy than ROH-bound states when the [4Fe-4S] cluster is 1e(-) reduced. Furthermore, η(2)-bound states can already be generated in the oxidized state, yielding reduction potentials of ca. -700 mV when electron addition occurs after rotation of the HMBPP C4-OH group. We demonstrate that such η(2)-bound states are kinetically accessible both when the IspH [4Fe-4S] cluster is oxidized and 1e(-) reduced. The energetically preferred pathway gives 1e(-) reduction of the cluster after substrate conformational change, generating the 1e(-) reduced intermediate proposed in the organometallic mechanism.