Activation of dioxygen at the carboxylate-bridged diiron(II) cluster in the R2 subunit of Escherichia coli class I ribonucleotide reductase produces the enzyme's catalytically essential stable tyrosyl radical by one-electron oxidation of tyrosine 122. An intermediate in the reaction, the formally Fe(IV)Fe(III) cluster X, can oxidize Y122 in the final and rate-limiting step. During formation of X, an “extra” electron must be transferred to an as-yet-uncharacterized adduct between O2 and the diiron(II) cluster. It was previously shown that a transient, broad absorption band centered near 560 nm develops when the reaction is carried out without an obvious exogenous source of the extra electron, and this band was ascribed to a tryptophan cation radical (W+•) resulting from temporary donation of the electron by the near-surface tryptophan residue 48 during formation of X [Bollinger, J. M., Jr.; Tong, W. H.; Ravi, N.; Huynh, B. H.; Edmondson, D. E.; Stubbe, J. J. Am. Chem. Soc. 1994, 116, 8024−8032]. In this work, we provide more definitive evidence for the W+• assignment by showing that (1) the absorbing species reacts rapidly with reductants, (2) the species is associated with a g = 2.0 EPR signal and perturbs the EPR and Mössbauer spectra of X, and (3) most definitively, the absorption spectrum of the species from 310 to 650 nm closely matches the very distinctive spectrum of the tryptophan cation radical previously determined in pulse radiolysis studies [Solar, S.; Getoff, N.; Surdhar, P. S.; Armstrong, D. A.; Sing, A. J. Phys. Chem. 1991, 95, 3639−3643]. Quantitation of species at short reaction times by optical, EPR, and Mössbauer spectroscopies is consistent with the rapid formation of an intermediate containing both X and the W+• (an X-W+• diradical species). Formation of the W+• (and presumably of X) is kinetically first order in both O2 and Fe(II)-R2 complex, even at the highest reactant concentrations examined, which give a formation rate constant approaching 200 s-1. This observation implies that precursors to the diradical species must not accumulate to greater than ∼10% of the initial Fe(II)-R2 reactant concentration and that the immediate precursor must generate the highly oxidizing W+• with a rate constant of at least 400 s-1 at 5 °C.

Plasmodium faliciparum causes 1-2 million deaths annually, yet current drug therapies are compromised by resistance. We previously described potent and selective triazolopyrimidine-based inhibitors of P. falciparum dihydroorotate dehydrogenase (PfDHODH) that inhibited parasite growth in vitro, however they showed no activity in vivo. Here we show that lack of efficacy against P. berghei in mice resulted from a combination of poor plasma exposure, and reduced potency against P. berghei DHODH. For compounds containing naphthyl (DSM1) or anthracenyl (DSM2), plasma exposure was reduced upon repeated dosing. Phenyl-substituted triazolopyrimidines were synthesized leading to identification of analogs with low predicted metabolism in human liver microsomes and which showed prolonged exposure in mice. Compound 21 (DSM74), containing p-trifluoromethylphenyl, suppressed growth of P. berghei in mice after oral administration. This study provides the first proof of concept that DHODH inhibitors can suppress Plasmodium growth in vivo, validating DHODH as a new target for anti-malarial chemotherapy.

A Plasmodium falciparum dihydroorotate dehydrogenase ( PfDHODH) inhibitor that is potent ( KI = 15 nM) and species-selective (>5000-fold over the human enzyme) was identified by high-throughput screening. The substituted triazolopyrimidine and its structural analogues were produced by an inexpensive three-step synthesis, and the series showed good association between PfDHODH inhibition and parasite toxicity. This study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial activity in whole cells (EC50 = 79 nM).

Non-heme diiron clusters occur in a number of enzymes (e.g., ribonucleotide reductase, methane monooxygenase, and Delta9-stearoyl-ACP desaturase) that activate O2 for chemically difficult oxidation reactions. In each case, a kinetically labile peroxo intermediate is believed to form when O2 reacts with the diferrous enzyme, followed by O-O bond cleavage and the formation of high-valent iron intermediates [formally Fe(IV)] that are thought to be the reactive oxidants. Greater kinetic stability of a peroxodiiron(III) intermediate in protein R2 of ribonucleotide reductase was achieved by the iron-ligand mutation Asp84 --> Glu and the surface mutation Trp48 --> Phe. Here, we present the first definitive evidence for a bridging, symmetrical peroxo adduct from vibrational spectroscopic studies of the freeze-trapped intermediate of this mutant R2. Isotope-sensitive bands are observed at 870, 499, and 458 cm-1 that are assigned to the intraligand peroxo stretching frequency and the asymmetric and symmetric Fe-O2-Fe stretching frequencies, respectively. Similar results have been obtained in the resonance Raman spectroscopic study of a peroxodiferric species of Delta9-stearoyl-ACP desaturase [Broadwater, J. A., Ai, J., Loehr, T. M., Sanders-Loehr, J., and Fox, B. G. (1998) Biochemistry 37, 14664-14671]. Similarities among these adducts and transient species detected during O2 activation by methane monooxygenase hydroxylase, ferritin, and wild-type protein R2 suggest the symmetrical peroxo adduct as a common intermediate in the diverse oxidation reactions mediated by members of this class.

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and ureabased compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC 50 values below 600 nM were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC 50 against malarial DHODH of 16 nM, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.

Analysis of the spectroscopic signatures of the R2-W48F/D84E biferric peroxo intermediate identifies a cis mu-1,2 peroxo coordination geometry. DFT geometry optimizations on both R2-W48F/D84E and R2-wild-type peroxo intermediate models including constraints imposed by the protein also identify the cis mu-1,2 peroxo geometry as the most stable peroxo intermediate structure. This study provides significant insight into the electronic structure and reactivity of the R2-W48F/D84E peroxo intermediate, structurally related cis mu-1,2 peroxo model complexes, and other enzymatic biferric peroxo intermediates.

The mechanism and outcome of dioxygen activation by the carboxylate-bridged diiron(II) cluster in the W48F site-directed variant of protein R2 of ribonucleotide reductase from Escherichia coli has been investigated by kinetic, spectroscopic, and chemical methods. The data corroborate the hypothesis advanced in earlier work and in the preceding paper that W48 mediates, by a shuttling mechanism in which it undergoes transient one-electron oxidation, the transfer of the "extra" electron that is required for formation of the formally Fe(IV)Fe(III) cluster X on the reaction pathway to the tyrosyl radical/µ-oxodiiron(III) cofactor of the catalytically active protein. The transient 560-nm absorption, which develops in the reaction of the wild-type R2 protein and is ascribed to the W48 cation radical, is not observed in the reaction of R2-W48F. Instead, a diradical intermediate containing both X and the Y122 radical (X-Y • ) accumulates rapidly to a high level. The formation of this X-Y • species is demonstrated indirectly by optical, Mo ¨ssbauer, and EPR kinetic data, which show concomitant accumulation of the two constituents, and directly by the unique EPR and Mo ¨ssbauer spectroscopic features of the X-Y • species, which can be properly simulated by using the known magnetic properties of X and Y122 • and introducing a spin-spin interaction between the two radicals. This analysis of the spectroscopic data provides an estimate of the distance between the two radical constituents that is consistent with the crystallographically defined distance between Y122 and the diiron cluster. These results suggest that substitution of W48 with phenylalanine impairs the pathway through which the extra electron is normally transferred. As a result, the two-electron-oxidized diiron species, designated as (Fe 2 O 2 ) 4+ , which in wild-type R2 would oxidize W48 to form X and the W48 +• , instead oxidizes Y122 to form the X-Y • . Most of the Y122 • that forms as part of the X-Y • subsequently decays. Decay of the Y122 • probably results from further reaction with the adjacent X, as indicated by the formation of altered diiron(III) products and by the ability of the strong reductant, dithionite, to "rescue" the Y122 • from decay by reducing X to form the normal µ-oxo diiron(III) cluster.The preceding paper presents evidence that a transient tryptophan cation radical (W +• ) forms upon reaction of O 2 with the diiron(II) cluster in protein R2 of E. coli class I ribonucleotide reductase (RNR). 1,2 The demonstration that this radical forms rapidly in a second-order reaction between the reactive Fe(II)-R2 complex and O 2 and is efficiently reduced by Fe-(II) aq , 2-mercaptoethanol, and ascorbate implicates a tryptophanmediated electron-shuttling mechanism for transfer of the extra electron that is required for formation of the formally Fe(IV)-Fe(III) intermediate, cluster X, on the reaction pathway that leads to the tyrosyl radical/µ-oxo-diiron(III) cofactor of catalytically active RNR. A hydrogen-bonded network comprising the near surface ...

Nuclear gamma resonance spectroscopy, also known as Mössbauer spectroscopy, is a technique that probes transitions between the nuclear ground state and a low-lying nuclear excited state. The nucleus most amenable to Mössbauer spectroscopy is 57Fe, and 57Fe Mössbauer spectroscopy provides detailed information about the chemical environment and electronic structure of iron. Iron is by far the most structurally and functionally diverse metal ion in biology, and 57Fe Mössbauer spectroscopy has played an important role in the elucidation of its biochemistry. In this article, we give a brief introduction to the technique and then focus on two recent exciting developments pertaining to the application of 57Fe Mössbauer spectroscopy in biochemistry. The first is the use of the rapid freeze-quench method in conjunction with Mössbauer spectroscopy to monitor changes at the Fe site during a biochemical reaction. This method has allowed for trapping and subsequent detailed spectroscopic characterization of reactive intermediates and thus has provided unique insight into the reaction mechanisms of Fe-containing enzymes. We outline the methodology using two examples: (1) oxygen activation by the non-heme diiron enzymes and (2) oxygen activation by taurine:alpha-ketoglutarate dioxygenase (TauD). The second development concerns the calculation of Mössbauer parameters using density functional theory (DFT) methods. By using the example of TauD, we show that comparison of experimental Mössbauer parameters with those obtained from calculations on model systems can be used to provide insight into the structure of a reaction intermediate.