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ABSTRACT: Herein, we describe the optimization of a linked enzyme assay suitable for high-throughput screening of decarboxylases, a target family whose activity has historically been difficult to quantify. Our approach uses a commercially available bicarbonate detection reagent to measure decarboxylase activity. The assay is performed in a fully enclosed automated screening system under inert nitrogen atmosphere to minimize perturbation by exogenous CO2. Receiver operating characteristic (ROC) analysis following a pilot screen of a small library of approximately 3,600 unique molecules for inhibitors of Trypanosoma brucei ornithine decarboxylase quantitatively demonstrates that the assay has excellent discriminatory power (area under the curve = 0.90 with 95% confidence interval between 0.82 and 0.97).
Assay and Drug Development Technologies 04/2010; 8(2):175-85. · 1.73 Impact Factor
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Ramesh Gujjar,
Alka Marwaha,
Farah El Mazouni,
John White,
Karen L White,
Sharon Creason,
David M Shackleford, Jeffrey Baldwin,
William N Charman,
Frederick S Buckner,
Susan Charman,
Pradipsinh K Rathod,
Margaret A Phillips
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ABSTRACT: Plasmodium falciparum 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 antimalarial chemotherapy.
Journal of Medicinal Chemistry 05/2009; 52(7):1864-72. · 4.80 Impact Factor
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ABSTRACT: 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).
Journal of Medicinal Chemistry 06/2008; 51(12):3649-53. · 5.25 Impact Factor
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ABSTRACT: The survival of the malaria parasite Plasmodium falciparum is dependent upon the de novo biosynthesis of pyrimidines. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in this pathway in an FMN-dependent reaction. The full-length enzyme is associated with the inner mitochondrial membrane, where ubiquinone (CoQ) serves as the terminal electron acceptor. The lipophilic nature of the co-substrate suggests that electron transfer to CoQ occurs at the two-dimensional lipid-solution interface. Here we show that PfDHODH associates with liposomes even in the absence of the N-terminal transmembrane-spanning domain. The association of a series of ubiquinone substrates with detergent micelles was studied by isothermal titration calorimetry, and the data reveal that CoQ analogs with long decyl (CoQ(D)) or geranyl (CoQ(2)) tails partition into detergent micelles, whereas that with a short prenyl tail (CoQ(1)) remains in solution. PfDHODH-catalyzed reduction of CoQ(D) and CoQ(2), but not CoQ(1), is stimulated as detergent concentrations (Tween 80 or Triton X-100) are increased up to their critical micelle concentrations, beyond which activity declines. Steady-state kinetic data acquired for the reaction with CoQ(D) and CoQ(2) in substrate-detergent mixed micelles fit well to a surface dilution kinetic model. In contrast, the data for CoQ(1) as a substrate were well described by solution steady-state kinetics. Our results suggest that the partitioning of lipophilic ubiquinone analogues into detergent micelles needs to be an important consideration in the kinetic analysis of enzymes that utilize these substrates.
Journal of Biological Chemistry 05/2007; 282(17):12678-86. · 4.77 Impact Factor
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ABSTRACT: The survival of the malaria parasite Plasmodium falciparum is dependent upon the de novo biosynthesis of pyrimidines. P. falciparum dihydroorotate dehydrogenase (PfDHODH) catalyzes the fourth step in this pathway in an FMN-dependent reaction. The full-length enzyme is associated with the
inner mitochondrial membrane, where ubiquinone (CoQ) serves as the terminal electron acceptor. The lipophilic nature of the
co-substrate suggests that electron transfer to CoQ occurs at the two-dimensional lipid-solution interface. Here we show that
PfDHODH associates with liposomes even in the absence of the N-terminal transmembrane-spanning domain. The association of a
series of ubiquinone substrates with detergent micelles was studied by isothermal titration calorimetry, and the data reveal
that CoQ analogs with long decyl (CoQD) or geranyl (CoQ2) tails partition into detergent micelles, whereas that with a short prenyl tail (CoQ1) remains in solution. PfDHODH-catalyzed reduction of CoQD and CoQ2, but not CoQ1, is stimulated as detergent concentrations (Tween 80 or Triton X-100) are increased up to their critical micelle concentrations,
beyond which activity declines. Steady-state kinetic data acquired for the reaction with CoQD and CoQ2 in substrate-detergent mixed micelles fit well to a surface dilution kinetic model. In contrast, the data for CoQ1 as a substrate were well described by solution steady-state kinetics. Our results suggest that the partitioning of lipophilic
ubiquinone analogues into detergent micelles needs to be an important consideration in the kinetic analysis of enzymes that
utilize these substrates.
Journal of Biological Chemistry 04/2007; 282(17):12678-12686. · 4.77 Impact Factor
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ABSTRACT: 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 urea-based 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.
Journal of Biological Chemistry 07/2005; 280(23):21847-53. · 4.77 Impact Factor
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ABSTRACT: 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.
Inorganic Chemistry 03/2005; 44(4):742-57. · 4.60 Impact Factor
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ABSTRACT: Ornithine decarboxylase (ODC) is an obligate homodimer that catalyzes the pyridoxal 5'-phosphate-dependent decarboxylation of l-ornithine to putrescine, a vital step in polyamine biosynthesis. A previous mutagenic analysis of the ODC dimer interface identified several residues that were distant from the active site yet had a greater impact on catalytic activity than on dimer stability [Myers, D. P., et al. (2001) Biochemistry 40, 13230-13236]. To better understand the basis of this phenomenon, the structure of the Trypanosoma brucei ODC mutant K294A was determined to 2.15 A resolution in complex with the substrate analogue d-ornithine. This residue is distant from the reactive center (>10 A from the PLP Schiff base), and its mutation reduced catalytic efficiency by 3 kcal/mol. The X-ray structure demonstrates that the mutation increases the disorder of residues Leu-166-Ala-172 (Lys-169 loop), which normally form interactions with Lys-294 across the dimer interface. In turn, the Lys-169 loop forms interactions with the active site, suggesting that the reduced catalytic efficiency is mediated by the decreased stability of this loop. The extent of disorder varies in the four Lys-169 loops in the asymmetric unit, suggesting that the mutation has led to an increase in the population of inactive conformations. The structure also reveals that the mutation has affected the nature of the ligand-bound species. Each of the four active sites contains unusual ligands. The electron density suggests one active site contains a gem-diamine intermediate with d-ornithine; the second has density consistent with a tetrahedral adduct with glycine, and the remaining two contain tetrahedral adducts of PLP, Lys-69, and water (or hydroxide). These data also suggest that the structure is less constrained in the mutant enzyme. The observation of a gem-diamine intermediate provides insight into the conformational changes that occur during the ODC catalytic cycle.
Biochemistry 11/2004; 43(41):12990-9. · 3.42 Impact Factor
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ABSTRACT: Paramecium bursaria chlorella virus (PBCV-1) is a large double-stranded DNA virus that infects chlorella-like green algae. The virus encodes a homolog of eukaryotic ornithine decarboxylase (ODC) that was previously demonstrated to be capable of decarboxylating l-ornithine. However, the active site of this enzyme contains a key amino acid substitution (Glu for Asp) of a residue that interacts with the delta-amino group of ornithine analogs in the x-ray structures of ODC. To determine whether this active-site change affects substrate specificity, kinetic analysis of the PBCV-1 decarboxylase (PBCV-1 DC) on three basic amino acids was undertaken. The k(cat)/K(m) for l-arginine is 550-fold higher than for either l-ornithine or l-lysine, which were decarboxylated with similar efficiency. In addition, alpha-difluoromethylarginine was a more potent inhibitor of the enzyme than alpha-difluoromethylornithine. Mass spectrometric analysis demonstrated that inactivation was consistent with the formation of a covalent adduct at Cys(347). These data demonstrate that PBCV-1 DC should be reclassified as an arginine decarboxylase. The eukaryotic ODCs, as well as PBCV-1 DC, are only distantly related to the bacterial and plant arginine decarboxylases from their common beta/alpha-fold class; thus, the finding that PBCV-1 DC prefers l-arginine to l-ornithine was unexpected based on evolutionary analysis. Mutational analysis was carried out to determine whether the Asp-to-Glu substitution at position 296 (position 332 in Trypanosoma brucei ODC) conferred the change in substrate specificity. This residue was found to be an important determinant of substrate binding for both l-arginine and l-ornithine, but it is not sufficient to encode the change in substrate preference.
Journal of Biological Chemistry 09/2004; 279(34):35760-7. · 4.77 Impact Factor
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ABSTRACT: 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.
Journal of the American Chemical Society 08/2004; 126(28):8842-55. · 9.91 Impact Factor
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ABSTRACT: Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*). A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X. Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine [Krebs, C., Chen, S., Baldwin, J., Ley, B. A., Patel, U., Edmondson, D. E., Huynh, B. H., and Bollinger, J. M., Jr. (2000) J. Am. Chem. Soc. 122, 12207-12219]. In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds. In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product. In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex. Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome. In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122*. In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus.
Biochemistry 06/2004; 43(20):5943-52. · 3.42 Impact Factor
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ABSTRACT: The R2 subunit of Escherichia coli ribonucleotide reductase contains a dinuclear iron center that generates a catalytically essential stable tyrosyl radical by one electron oxidation of a nearby tyrosine residue. After acquisition of Fe(II) ions by the apo protein, the resulting diiron(II) center reacts with O(2) to initiate formation of the radical. Knowledge of the structure of the reactant diiron(II) form of R2 is a prerequisite for a detailed understanding of the O(2) activation mechanism. Whereas kinetic and spectroscopic studies of the reaction have generally been conducted at pH 7.6 with reactant produced by the addition of Fe(II) ions to the apo protein, the available crystal structures of diferrous R2 have been obtained by chemical or photoreduction of the oxidized diiron(III) protein at pH 5-6. To address this discrepancy, we have generated the diiron(II) states of wildtype R2 (R2-wt), R2-D84E, and R2-D84E/W48F by infusion of Fe(II) ions into crystals of the apo proteins at neutral pH. The structures of diferrous R2-wt and R2-D48E determined from these crystals reveal diiron(II) centers with active site geometries that differ significantly from those observed in either chemically or photoreduced crystals. Structures of R2-wt and R2-D48E/W48F determined at both neutral and low pH are very similar, suggesting that the differences are not due solely to pH effects. The structures of these "ferrous soaked" forms are more consistent with circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopic data and provide alternate starting points for consideration of possible O(2) activation mechanisms.
Journal of the American Chemical Society 01/2004; 125(51):15822-30. · 9.91 Impact Factor
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ABSTRACT: The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by Mössbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.
Biochemistry 12/2003; 42(45):13269-79. · 3.42 Impact Factor
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ABSTRACT: The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm. The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.
Journal of Biological Chemistry 12/2002; 277(44):41827-34. · 4.77 Impact Factor
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ABSTRACT: The malarial parasite relies onde novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed
pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key
step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite.
To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k
cat 8 s−1, K
DHO (40–80 μm)). The substrate specificity of the ubiquinone cofactor ( CoQn) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length
of CoQn was a determinant of reaction efficiency; CoQ4, CoQ6 and decylubiquinone ( CoQD) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ0 and vitamin K3) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K
. Five potent inhibitors of mammalian DHODH, Redoxal , dichloroallyl lawsone ( DCL ), and three analogs of A77 1726 were
tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC50's ranging from 0.1–1.0 mm. The IC50 values for inhibition of the malarial enzyme are 102-104-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific.
These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is
an attractive target for the development of new anti-malarial agents.
Journal of Biological Chemistry 10/2002; 277(44):41827-41834. · 4.77 Impact Factor
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ABSTRACT: 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, Mössbauer, and EPR kinetic data, which show concomitant accumulation of the two constituents, and directly by the unique EPR and Mössbauer 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 (Fe2O2)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.
11/2000;
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ABSTRACT: 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.
11/2000;
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ABSTRACT: Ribonucleotide reductase (RR) catalyzes the first committed and rate-determining step in DNA biosynthesis, the reduction of ribonucleotides to deoxyribonucleotides. FeII binding to the binuclear non-heme iron active site has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD spectroscopies. These studies show that the two sites have significantly different metal binding affinities. This has also allowed a MnIIFeII derivative to be prepared and studied by the above spectroscopies. The spectral features of the individual irons provide geometric and electronic structural insight into each metal site. Density functional calculations on reduced RR are correlated to the spectral features to obtain insight into its electronic structure. Parallel calculations are also performed on reduced stearoyl-acyl carrier protein Δ9 desaturase (Δ9D) to correlate to prior spectral data and to the active site of RR. Differences in their dioxygen reactivities are investigated through reaction of these reduced sites with dioxygen, and possible electron-transfer pathways are evaluated. These results show that the active site of reduced RR consists of one 5- and one 4-coordinate iron with the 5C center having a higher binding affinity. Compared to reduced Δ9D, the presence of the 4C site energetically destabilizes reduced RR. Reaction of reduced RR with dioxygen to form a superoxide intermediate is energetically up hill as it results in an excited quartet state on the oxygenated iron, while the formation of a bridged peroxo intermediate is energetically favorable. Formation of peroxo-RR is more favorable than peroxo-Δ9D due to ligand field differences that can control the overlap of the redox active orbitals of the reduced sites with the π* orbitals of dioxygen. This parallels experimental differences in the dioxygen reactivity of the reduced RR and Δ9D active sites.
08/2000;
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ABSTRACT: The R2 subunit of Escherichia coli (aerobic) ribonucleotide reductase activates molecular oxygen at its diiron center to produce a functionally essential stable tyrosyl radical from residue Y122. It was previously shown that the D84E site-directed mutant of R2 (R2-D84E) accumulates a μ-1,2-peroxodiiron(III) intermediate on the pathway to tyrosyl radical formation. This intermediate does not accumulate in the reaction of wildtype (wt) R2, but an analogous complex does accumulate during oxygen activation by the structurally similar diiron protein, methane monooxygenase hydroxylase (MMOH). Herein we describe the crystallographically determined three-dimensional structures of the reduced, diiron(II) reactant and oxidized, diiron(III) product forms of R2-D84E. The reduced R2-D84E structure differs from that of reduced wt R2 in the conformations of three carboxylate ligands, E84, E204, and E238. The adjustments in these ligands render the coordination sphere of the diiron(II) center very similar to that in reduced MMOH. In addition, a water molecule not observed in reduced wt R2 is coordinated to Fe2 in reduced R2-D84E. The oxidized R2-D84E structure is similar to that of oxidized wt R2 except in the coordination mode of E84. In R2-D84E, E84 coordinates to Fe1 in a monodentate, terminal mode and is hydrogen bonded to a water molecule also coordinated to Fe1. In wt R2, D84 is a bidentate, chelating ligand. In both R2-D84E structures, Y122 is shifted away from Fe1 such that a hydrogen bonding interaction with E84 is not possible. The observed structural adjustments suggest possible rationales for the stability of the μ-1,2-peroxodiiron(III) complex in R2-D84E. In addition, the structures expand the experimental foundation for computational investigations aimed at defining the detailed mechanistic pathways for O2 activation at diiron(II) centers.
03/2000;
