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 π∕σ "metallacycle" or η 2 -alkenyl complex. The complex is poised to interact with H þ 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 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.enzyme inhibition | iron-sulfur protein | isoprenoid biosynthesis | nonmevalonate pathway E nzymes that catalyze the formation of isoprenoids are of interest as drug targets. There are two main pathways involved in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococcus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative agents of staph infections, Chagas' disease and the leishmaniases), and the nonmevalonate or Rohmer pathway found in most pathogenic bacteria, as well as in the malaria parasite, Plasmodium falciparum (1). Both pathways lead to formation of the C 5 -isoprenoids isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2). In the later stages of isoprenoid biosynthesis, these C 5 -compounds then form the farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) used in protein prenylation, sterol, and carotenoid biosynthesis. Understanding how the enzymes catalyzing these "downstream" events function has led to a better understanding of e.g. how FPP synthase (2) and GGPP synthase function, and can be inhibited (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase and oxidosqualene cyclase inhibitor) against Chagas' disease (5; 6) and leishmaniasis (7); anticancer agents that inhibit both FPPS and GGPPS (8); as wel...

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe 4 S 4 cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using 2 H, 13 C and 17 O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η 2 -alkenyl π-(or π∕σ) complex with the 4th Fe in the Fe 4 S 4 cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K i ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13 C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π∕σ metallacycle, an η 2 -alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.4Fe-4S protein | GcpE (IspG) | metallacycle

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and 2H, 17O, and 57Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin: thioredoxin reductase, in that one electron is transferred to the [4Fe-4S]2+ cluster, which then performs a formally two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate’s 3-CH2OH group has rotated away from the reduced iron-sulfur cluster, and the next, η3-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogs (4 and 5) follow the same reaction mechanism.

We report the inhibition of the Aquifex aeolicus IspH enzyme (LytB, (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate reductase, EC 1.17.1.2) by a series of diphosphates and bisphosphonates. The most active species was an alkynyl diphosphate having an IC 50 = 0.45 μM (K i ~ 60 nM), which generated a very large change in the 9 GHz EPR spectrum of the reduced protein. Based on previous work on organometallic complexes, together with computational docking and quantum chemical calculations, we propose a model for alkyne inhibition involving π (or π/σ) "metallacycle" complex formation with the unique 4 th Fe in the Fe 4 S 4 cluster. Aromatic species had less activity and for these, we propose an inhibition model based on an electrostatic interaction with the active site E126. Overall, the results are of broad general interest since not only do they represent the first potent IspH inhibitors, they suggest a conceptually new approach to targeting other Fe 4 S 4 -cluster containing proteins that are of interest as drug and herbicide targets.

The methylerythritol phosphate pathway of isoprenoid biosynthesis is an attractive anti-infective drug target. The last two enzymes of this pathway, IspG and IspH, are [Fe4S4] proteins not produced by humans that catalyze 2H+/2e− reductions with novel mechanisms. In this review, we summarize recent advances in structural, mechanistic and inhibitory studies of these two enzymes. In particular, mechanistic proposals involving bioorganometallic intermediates are presented and compared with other mechanistic possibilities, and inhibitors based on substrate analogs, developed by rational design and compound library screening, are discussed. These results represent the first examples of bioorganometallic catalytic mechanisms of [Fe4S4] enzymes, and open up new routes to inhibitor design targeting [Fe4S4] clusters.

IspG is a 4Fe-4S protein that carries out an essential reduction step in isoprenoid biosynthesis. Using electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies on labeled samples, we specifically assign the hyperfine interactions in a reaction intermediate. These results help clarify the nature of the reaction intermediate, supporting a direct interaction between the unique 4th Fe in the cluster and the C2 and O3 of the ligand.

IspG is a 4Fe4S protein involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites, there is a large insert domain (A*) whose structure and function are unknown. We show that bacterial IspGs function in solution as (AB) 2 dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, because there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer. We propose, using computational modeling and electron microscopy, that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product, and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest because they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design.

The regulatory component (MMOB) of soluble methane monooxygenase (sMMO) has a unique N-terminal tail not found in regulatory proteins of other bacterial multicomponent monooxygenases. This N-terminal tail is indispensable for proper function, yet its solution structure and role in catalysis remain elusive. Here, by using double electron–electron resonance (DEER) spectroscopy, we show that the oxidation state of the hydroxylase component, MMOH, modulates the conformation of the N-terminal tail in the MMOH–2MMOB complex, which in turn facilitates catalysis. The results reveal that the N-terminal tail switches from a relaxed, flexible conformational state to an ordered state upon MMOH reduction from the diiron(III) to the diiron(II) state. This observation suggests that some of the crystallographically observed allosteric effects that result in the connection of substrate ingress cavities in the MMOH–2MMOB complex may not occur in solution in the diiron(III) state. Thus, O2 may not have easy access to the active site until after reduction of the diiron center. The observed conformational change is also consistent with a higher binding affinity of MMOB to MMOH in the diiron(II) state, which may allow MMOB to displace more readily the reductase component (MMOR) from MMOH following reduction.