Ferritin reactions: direct identification of the site for the diferric peroxide reaction intermediate

Ferritins managing iron-oxygen biochemistry in animals, plants, and microorganisms belong to the diiron carboxylate protein family and concentrate iron as ferric oxide approximately 10(14) times above the ferric K(s). Ferritin iron (up to 4,500 atoms), used for iron cofactors and heme, or to trap DNA-damaging oxidants in microorganisms, is concentrated in the protein nanocage cavity (5-8 nm) formed during assembly of polypeptide subunits, 24 in maxiferritins and 12 in miniferritins/DNA protection during starvation proteins. Direct identification of ferritin ferroxidase (F(ox)) sites, complicated by multiple types of iron-ferritin interactions, is now achieved with chimeric proteins where putative F(ox) site residues were introduced singly and cumulatively into an inactive host, an L maxiferritin. A dimagnesium ferritin cocrystal model guided site design and the diferric peroxo F(ox) intermediates (A at 650 nm) monitored activity. Diferric peroxo formation in chimeric and WT proteins had similar K(app) values and Hill coefficients. Catalytic activity required cooperative ferrous substrate binding to two sites A (E, EXXH) and B (E, QXXD). The weaker B sites in ferritin contrast with stronger B sites (E, EXXH) in diiron carboxylate oxygenases, explaining diferric oxo/hydroxo product release in ferritin vs. diiron cofactor retention in oxygenases. Codons for Q/H and D/E differ by single nucleotides, suggesting simple DNA mutations relate site B diiron substrate sites and diiron cofactor sites in proteins. The smaller k(cat) values in chimeras indicate the absence of second-shell residues important for ferritin substrate-product channeling that, when identified, will outline the entire iron path from ferritin pores through the F(ox) site to the mineral cavity.     

Carbon dioxide reduction to methane and coupling with acetylene to form propylene catalyzed by remodeled nitrogenase

A doubly substituted form of the nitrogenase MoFe protein (α-70^Val→Ala, α-195^His→Gln) has the capacity to catalyze the reduction of carbon dioxide (CO₂) to yield methane (CH₄). Under optimized conditions, 1 nmol of the substituted MoFe protein catalyzes the formation of 21 nmol of CH₄ within 20 min. The catalytic rate depends on the partial pressure of CO₂ (or concentration of HCO₃⁻) and the electron flux through nitrogenase. The doubly substituted MoFe protein also has the capacity to catalyze the unprecedented formation of propylene (H₂C = CH-CH₃) through the reductive coupling of CO₂ and acetylene (HC≡CH). In light of these observations, we suggest that an emerging understanding of the mechanistic features of nitrogenase could be relevant to the design of synthetic catalysts for CO₂ sequestration and formation of olefins.     

Low Temperature Electronic Absorption Spectra of Oxidized and Reduced Spinach Ferredoxins. Evidence for Nonequivalent Iron(III) Sites

The electronic absorption spectra of oxidized and reduced spinach ferredoxins have been measured between 1200 and 600 nm at low temperature in D₂O/ethylene glycol glasses. Relatively weak absorption bands are observed at 720, 820, and 920 nm in oxidized ferredoxin, and at 652, 820, and 920 nm in reduced ferredoxin. The spectral results show that the two Fe(III) centers in oxidized ferredoxin are not equivalent, and that the 820- and 920-nm bands are associated with the nonreducible site. Assignment of the reducible site as tetrahedral Fe(III) is indicated. The 720-nm (13.9 kcm^-1) band in oxidized ferredoxin is attributed to an intensity-enhanced ⁶A₁ → ⁴T₁d-d transition, whereas the 652-nm (15.3 kcm^-1) feature of reduced ferredoxin could be due either to ⁵E → ³T₁ in tetrahedral Fe(II)S₄ or an Fe(II) → Fe(III) intervalence excitation.     

Substrate positioning controls the partition between halogenation and hydroxylation in the aliphatic halogenase,SyrB2

The α-ketoglutarate-dependent hydroxylases and halogenases employ similar reaction mechanisms involving hydrogen-abstracting Fe(IV)-oxo (ferryl) intermediates. In the halogenases, the carboxylate residue from the His₂(Asp/Glu)₁“facial triad” of iron ligands found in the hydroxylases is replaced by alanine, and a halide ion (X⁻) coordinates at the vacated site. Halogenation is thought to result from “rebound” of the halogen radical from the X-Fe(III)-OH intermediate produced by hydrogen (H^•) abstraction to the substrate radical. The alternative decay pathway for the X-Fe(III)-OH intermediate, rebound of the hydroxyl radical to the substrate radical (as occurs in the hydroxylases), reportedly does not compete. Here we show for the halogenase SyrB2 that positioning of the alkyl group of the substrate away from the oxo/hydroxo ligand and closer to the halogen ligand sacrifices H^•-abstraction proficiency for halogen-rebound selectivity. Upon replacement of l-Thr, the C4 amino acid tethered to the SyrB1 carrier protein in the native substrate, by the C5 amino acid l-norvaline, decay of the chloroferryl intermediate becomes 130× faster and the reaction outcome switches to primarily hydroxylation of C5, consistent with projection of the methyl group closer to the oxo/hydroxo by the longer side chain. Competing H^• abstraction from C4 results primarily in chlorination, as occurs at this site in the native substrate. Consequently, deuteration of C5, which slows attack at this site, switches both the regioselectivity from C5 to C4 and the chemoselectivity from hydroxylation to chlorination. Thus, substrate-intermediate disposition and the carboxylate → halide ligand swap combine to specify the halogenation outcome.     

Direct observation of structurally encoded metal discrimination and ether bond formation in a heterodinuclear metalloprotein

Although metallocofactors are ubiquitous in enzyme catalysis, how metal binding specificity arises remains poorly understood, especially in the case of metals with similar primary ligand preferences such as manganese and iron. The biochemical selection of manganese over iron presents a particularly intricate problem because manganese is generally present in cells at a lower concentration than iron, while also having a lower predicted complex stability according to the Irving–Williams series (Mn^II < Fe^II < Ni^II < Co^II < Cu^II > Zn^II). Here we show that a heterodinuclear Mn/Fe cofactor with the same primary protein ligands in both metal sites self-assembles from Mn^II and Fe^II in vitro, thus diverging from the Irving–Williams series without requiring auxiliary factors such as metallochaperones. Crystallographic, spectroscopic, and computational data demonstrate that one of the two metal sites preferentially binds Fe^II over Mn^II as expected, whereas the other site is nonspecific, binding equal amounts of both metals in the absence of oxygen. Oxygen exposure results in further accumulation of the Mn/Fe cofactor, indicating that cofactor assembly is at least a two-step process governed by both the intrinsic metal specificity of the protein scaffold and additional effects exerted during oxygen binding or activation. We further show that the mixed-metal cofactor catalyzes a two-electron oxidation of the protein scaffold, yielding a tyrosine–valine ether cross-link. Theoretical modeling of the reaction by density functional theory suggests a multistep mechanism including a valyl radical intermediate.Half of all enzymes are estimated to contain metallocofactors (1). An important subset uses transition metal ions to perform key redox reactions such as oxygen activation. The diiron cofactor of the ferritin-like superfamily of proteins is particularly versatile (2). While ferritin itself simply oxidizes and sequesters iron (3), in other family members the diiron center acts as a transient one- or two-electron oxidant. In the R2 subunits of class I ribonucleotide reductases (RNRs) it generates a redox-active tyrosyl radical (4, 5), whereas in the bacterial multicomponent monooxygenases (BMMs) it catalyzes the hydroxylation of a variety of hydrocarbons (6). For four decades it was assumed that all ferritin superfamily proteins contained diiron cofactors. However, in recent years new subfamilies harboring either a dimanganese or heterodinuclear Mn/Fe cofactor have been documented (7–14). The Mn/Fe cofactor was discovered in class Ic RNR R2 subunits, where its Mn^IV/Fe^III state functionally replaces the diiron-tyrosyl radical cofactor of class Ia R2s (9, 10). After a long controversy, class Ib R2 proteins were shown to use a dimanganese cofactor in the same scaffold (7, 8). These recent developments highlight the complexity of correctly identifying the metals that make up native metallocofactors. While the metal preferences of some primary coordination motifs are well known and distinct, others are more promiscuous and less well understood. Manganese and iron are two of the most important and versatile metals for biological redox chemistry. Their primary ligand preferences are very similar, and their binding sites in enzymes often appear virtually identical (15). However, their redox potentials differ greatly, and correct discrimination between them is therefore paramount for redox-active enzymes (16). Metal specificity is commonly discussed in terms of the intracellular availability of metal ions and the Irving–Williams series of metal complex stabilities (Mn^II < Fe^II < Ni^II < Co^II < Cu^II > Zn^II) (17). Manganese is generally present in cells at a lower concentration than iron (18) and also has a lower predicted complex stability. The biochemical selection of manganese over iron thus presents an intricate problem. Metallochaperones are required for correct metallation of some metalloproteins (19–22), but to date no chaperones have been identified for manganese, and it is unclear whether they are required for diiron clusters (16). Protein-folding location or general metal status can also control metallation (16, 23). However, these mechanisms cannot be used for proteins with mixed-metal cofactors.The heterodinuclear Mn/Fe cofactor recently discovered in a protein from Mycobacterium tuberculosis provides an ideal model system to study manganese/iron metallation. The protein was found to belong to a novel group of R2-like proteins, denoted R2-like ligand-binding oxidases (R2lox) (11, 24). These proteins are particularly interesting because the primary protein-derived metal-binding ligands are identical in both metal sites, and also identical to the closely related diiron-binding BMMs. To verify that the mixed-metal cofactor is a common feature of the R2lox group, here we characterize a homolog from the thermophilic bacterium Geobacillus kaustophilus (GkR2loxI). Using crystallography, spectroscopy, and quantum chemical calculations, we show that site-specific metal discrimination is inherent to the protein structure and diverges from the Irving–Williams series without requiring auxiliary factors in vitro. The mixed-metal cofactor activates oxygen and catalyzes the formation of an ether cross-link in the protein scaffold, demonstrating the chemical potential of this cofactor.     

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly¹³⁷ → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp¹³⁷ substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp¹³⁷ in the homologous position in acetylcholinesterase suggests that Asp¹³⁷ may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.     

Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates

Ferritin biominerals are protein-caged metabolic iron concentrates used for iron–protein cofactors and oxidant protection (Fe²⁺ and O₂ sequestration). Fe²⁺ passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe²⁺ substrate movement to ferritin enzyme (F_ox) sites. Fe²⁺ and O₂ substrates are coupled via a diferric peroxo (DFP) intermediate, λ_max 650 nm, which decays to [Fe³⁺–O–Fe³⁺] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (K_cat and k_cat/K_m), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe²⁺ concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe²⁺ access), Mn²⁺ << Co²⁺ < Cu²⁺ < Zn²⁺, reflecting metal ion–protein binding stabilities. Fe²⁺–Cys126 binding in ferritin ion channels, observed as Cu²⁺–S–Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu²⁺ inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe²⁺ movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe²⁺ into ferritin enzymatic sites. The results clarify Fe²⁺ transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.Biological and synthetic ion channels transport metal ions through barriers, including those in ferritin protein cages (1–8). How ions reach specific destinations, after exiting ion channels, remains unknown. Ion channels embedded in soluble ferritin protein cages are more accessible to study than membrane ion channels embedded in lipid-rich membranes. Fe²⁺ substrate is transported into ferritin protein cages through multiple ion channels that penetrate the cages (Fig. 1 A–D) and is delivered to multiple ferritin enzyme (F_ox) sites. Each ferritin enzyme site binds 2 Fe²⁺ substrate ions which, in eukaroytic ferritins, react with O₂ substrate; the intermediate of enzyme activity is diferric peroxo (DFP) that decays to diferric oxy, the precursors of protein-caged ferritin biominerals (Fe₂O₃⋅H₂O). Ferritin biominerals vary in size, depending on iron availability, up to 4,500 Fe/cage. Rapid (ms) formation of blue color DFP (λ_max: 650 nm) (9, 10) requires cooperative binding of 2 Fe²⁺/active site (Hill coefficient = 3) (11) and indicates correct Fe²⁺ delivery to the multiple ferritin enzymatic centers. Ferritin protein-caged iron biominerals, slow to dissolve in solution (Fig. S1), are metabolic iron concentrates for cofactor biosynthesis such as heme or iron-sulfur clusters. The conversion of Fe²⁺ and O₂ into protein-coated solids inside ferritin cages is an antioxidant activity, and ferritin genes are regulated with other antioxidant response proteins (4, 12, 13).Open in a separate windowFig. 1.Ion channels in soluble ferritin protein nanocages transport Fe²⁺ to multiple catalytic centers (F_ox or oxidoreductase sites). (A) The 24-subunit ferritin protein cage. Red helices, Fe²⁺ channels. (B) A ferritin Fe²⁺ ion channel (side view). Green spheres, metal ions. Conserved residues E57 and E136 are putative carboxylate links in Fe²⁺ transit between ferritin ion channel carboxylate (E130 and D127) and ferritin enzyme (F_ox) sites based on phylogenetic structural location and conformational flexibility in protein crystals (16, 18). (C) A single ferritin protein cage subunit (side view). Tan, channel helix segments. (D) Ion channel (viewed from inside the protein cage). Green spheres, metal ions. Prepared from PDB 3KA3 using PyMol.Ferritin protein ion channels are 15 Å long, analogous to short ion channels in biological membranes (3). Positioned around the threefold symmetry axes of the cage (Fig. 1), ferritin protein ion channels form during cage self-assembly from three proximal subunits; ion channel amino acids are highly conserved (14). Both external and internal ferritin ion channel pores are 4–5 Å in diameter with a midchannel constriction, 2.3 Å in diameter, created by the carboxylate side chains from the three glutamate residues at position 130. In protein crystals, ferritin ion channels bind a variety of divalent metal ions (Fig. S2B and Table S1). Because hydrated ions have diameters of 6.4–6.9 Ǻ, partial dehydration must occur for ion passage through the E130 constriction (2.3-Å diameter). Only single metal atoms are observed in protein–metal crystal structures at the ring of three E130 residues, contrasting with the multiple metal atoms accumulated near the channel exits around the ring of three D127 residues with shorter carboxylate side chains (Fig. 1 B and C); multiple ordered water molecules also accumulate near D127.

Table 1.

Conservation of ferritin amino acids in Fe²⁺ cage transfer residues and ion channel walls

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P₂) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P₂ decreased mating, from 2-fold for Arg to >10⁶-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (k_cat/K_M) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that K_M, which is heavily influenced by the nature of the P₂ residue, is a major determinant of cleavage efficiency in vivo. P₂ preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.     

Probing of the rates of alternating access in LacY with Trp fluorescence

Sugar/H⁺ symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H⁺ binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK_a of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is ≈100 s⁻¹. On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140→Trp/Phe-334→His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK_a = 8), but remarkably, the rate of sugar-induced quenching is only ≈0.4 s⁻¹. The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.     

Insights into the P-to-Q conversion in the catalytic cycle of methane monooxygenase from a synthetic model system

For the catalytic cycle of soluble methane monooxygenase (sMMO), it has been proposed that cleavage of the O–O bond in the (μ-peroxo)diiron(III) intermediate P gives rise to the diiron(IV) intermediate Q with an Fe₂(μ–O)₂ diamond core, which oxidizes methane to methanol. As a model for this conversion, (μ–oxo) diiron(III) complex 1 ([Fe^III₂(μ–O)(μ–O₂H₃)(L)₂]³⁺, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) has been treated consecutively with one eq of H₂O₂ and one eq of HClO₄ to form 3 ([Fe^IV₂(μ–O)₂(L)₂]⁴⁺). In the course of this reaction a new species, 2, can be observed before the protonation step; 2 gives rise to a cationic peak cluster by ESI-MS at m/z 1,399, corresponding to the {[Fe₂O₃L₂H](OTf)₂}⁺ ion in which 1 oxygen atom derives from 1 and the other two originate from H₂O₂. Mössbauer studies of 2 reveal the presence of two distinct, exchange coupled iron(IV) centers, and EXAFS fits indicate a short Fe–O bond at 1.66 Å and an Fe–Fe distance of 3.32 Å. Taken together, the spectroscopic data point to an HO-Fe^IV-O-Fe^IV = O core for 2. Protonation of 2 results in the loss of H₂O and the formation of 3. Isotope labeling experiments show that the [Fe^IV₂(μ–O)₂] core of 3 can incorporate both oxygen atoms from H₂O₂. The reactions described here serve as the only biomimetic precedent for the conversion of intermediates P to Q in the sMMO reaction cycle and shed light on how a peroxodiiron(III) unit can transform into an [Fe^IV₂(μ–O)₂] core.     

Visualizing the substrate-, superoxo-, alkylperoxo-, and product-bound states at the nonheme Fe(II) site of homogentisate dioxygenase

Homogentisate 1,2-dioxygenase (HGDO) uses a mononuclear nonheme Fe²⁺ to catalyze the oxidative ring cleavage in the degradation of Tyr and Phe by producing maleylacetoacetate from homogentisate (2,5-dihydroxyphenylacetate). Here, we report three crystal structures of HGDO, revealing five different steps in its reaction cycle at 1.7–1.98 Å resolution. The resting state structure displays an octahedral coordination for Fe²⁺ with two histidine residues (His331 and His367), a bidentate carboxylate ligand (Glu337), and two water molecules. Homogentisate binds as a monodentate ligand to Fe²⁺, and its interaction with Tyr346 invokes the folding of a loop over the active site, effectively shielding it from solvent. Binding of homogentisate is driven by enthalpy and is entropically disfavored as shown by anoxic isothermal titration calorimetry. Three different reaction cycle intermediates have been trapped in different HGDO subunits of a single crystal showing the influence of crystal packing interactions on the course of enzymatic reactions. The observed superoxo:semiquinone-, alkylperoxo-, and product-bound intermediates have been resolved in a crystal grown anoxically with homogentisate, which was subsequently incubated with dioxygen. We demonstrate that, despite different folds, active site architectures, and Fe²⁺ coordination, extradiol dioxygenases can proceed through the same principal reaction intermediates to catalyze the O₂-dependent cleavage of aromatic rings. Thus, convergent evolution of nonhomologous enzymes using the 2-His-1-carboxylate facial triad motif developed different solutions to stabilize closely related intermediates in unlike environments.     

Association of β2-adrenoceptor Gln27Glu variant with body weight but not hypertension

β₂-Adrenoceptor (β₂-ADR)-mediated vasodilatation decreases vascular reactivity and blood pressure (BP) and chromosome 5 where its gene (ADRB2R) resides and shows linkage to hypertension (HT). A Gln27Glu ADRB2R variant confers resistance to agonist-induced desensitization and enhanced vasodilator response to isoprenaline. Therefore, we carried out a case-control study in a cohort of HT and normotensive (NT) Anglo-Celtic Australian white subjects whose parents had a similar BP status as the subjects. Glu27 frequency was 0.41 in 108 HT and 0.42 in 141 NT (χ² = 0.05, P = .82). Within the HT group, the Glu27 allele was more prevalent in 61 subjects who were overweight (body mass index [BMI] ≥ 25 kg/m²) compared with 41 who were lean (BMI <25 kg/m²); ie, 0.49 v 0.31, respectively (χ² = 6.4, P = .012). Furthermore, Glu27 tracked with elevation in BMI in these subjects: 24 ± 4 kg/m², 27 ± 5 kg/m², and 28 ± 5 kg/m² for Gln/Gln, Gln/Glu, and Glu/Glu, respectively (P = .0058 by one-way ANOVA). Thus, the Gln27Glu β₂-ADR variant is excluded in HT, but might influence body weight.     

Phytochelatins, the heavy-metal-binding peptides of plants, are synthesized from glutathione by a specific gamma-glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase)

An enzyme has been discovered and characterized from Silene cucubalus cell suspension cultures that catalyzes the transfer of the γ-glutamylcysteine dipeptide moiety of glutathione to an acceptor glutathione molecule or a growing chain of [Glu(-Cys)]_n-Gly oligomers, thus synthesizing phytochelatins, the metal-binding peptides of higher plants and select fungi. The enzyme was named γ-glutamylcysteine dipeptidyl transpeptidase and given the trivial name phytochelatin synthase. The primary reaction catalyzed is [Glu(-Cys)]-Gly + [Glu(-Cys)]_n-Gly → [Glu(-Cys)]_n+1-Gly + Gly. The enzyme is isoelectric near pH 4.8 and has temperature and pH optima at 35°C and 7.9, respectively. Phytochelatin synthase is constitutively present in cell cultures of various plant species and its formation is not noticeably induced by heavy metal ions in the growth medium. The enzyme (M_r95,000) seems to be composed of four subunits, the dimer (M_r50,000) being also catalytically active. Cd²⁺ is by far the best metal activator of the enzyme followed by Ag⁺, Bi³⁺, Pb²⁺, Zn²⁺, Cu²⁺, Hg²⁺, and Au⁺. The K_m for glutathione is 6.7 mM. The enzyme activity seems to be self-regulated in that the product of the reaction (the phytochelatins) chelates the enzyme-activating metal, thus terminating the enzyme reaction. The molar ratio of the γ-glutamylcysteine dipeptide in phytochelatin to Cd²⁺ in the newly formed complex was 2:1.     

Engineering a model protein cavity to catalyze the Kemp elimination

Synthetic cavitands and protein cavities have been widely studied as models for ligand recognition. Here we investigate the Met102 → His substitution in the artificial L99A cavity in T4 lysozyme as a Kemp eliminase. The resulting enzyme had k_cat/K_M = 0.43 M^-1 s^-1 and a (k_cat/K_M)/k_uncat = 10⁷ at pH 5.0. The crystal structure of this enzyme was determined at 1.30 Å, as were the structures of four complexes of substrate and product analogs. The absence of ordered waters or hydrogen bonding interactions, and the presence of a common catalytic base (His102) in an otherwise hydrophobic, buried cavity, facilitated detailed analysis of the reaction mechanism and its optimization. Subsequent substitutions increased eliminase activity by an additional four-fold. As activity-enhancing substitutions were engineered into the cavity, protein stability decreased, consistent with the stability-function trade-off hypothesis. This and related model cavities may provide templates for studying protein design principles in radically simplified environments.     

A First-Principles Study of the Cu-Containing β″ Precipitates in Al-Mg-Si-Cu Alloy

The nanostructured β″ precipitates are critical for the strength of Al-Mg-Si-(Cu) aluminum alloys. However, there are still controversial reports about the composition of Cu-containing β″ phases. In this work, first-principles calculations based on density functional theory were used to investigate the composition, mechanical properties, and electronic structure of Cu-containing β″ phases. The results predict that the Cu-containing β″ precipitates with a stoichiometry of Mg_4+xAl_2−xCuSi₄ (x = 0, 1) are energetically favorable. As the concentration of Cu atoms increases, Cu-containing β″ phases with different compositions will appear, such as Mg₄AlCu₂Si₄ and Mg₄Cu₃Si₄. The replacement order of Cu atoms in β″ phases can be summarized as one Si3/Al site → two Si3/Al sites → two Si3/Al sites and one Mg1 site. The calculated elastic constants of the considered β″ phases suggest that they are all mechanically stable, and all β″ phases are ductile. When Cu atoms replace Al atoms at Si3/Al sites in β″ phases, the values of bulk modulus (B), shear modulus (G), and Young’s modulus (E) all increase. The calculation of the phonon spectrum shows that Mg_4+xAl_2−xCuSi₄ (x = 0, 1) are also dynamically stable. The electronic structure analysis shows that the bond between the Si atom and the Cu atom has a covalent like property. The incorporation of the Cu atom enhances the electron interaction between the Mg2 and the Si3 atom so that the Mg2 atom also joins the Si network, which may be one of the reasons why Cu atoms increase the structure stability of the β″ phases.     

Stearoyl-acyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein.

A gene encoding stearoyl-acyl carrier protein delta 9 desaturase (EC 1.14.99.6) from castor was expressed in Escherichia coli. The purified catalytically active enzyme contained four atoms of iron per homodimer. The desaturase was studied in two oxidation states with Mössbauer spectroscopy in applied fields up to 6.0 T. These studies show conclusively that the oxidized enzyme contains two (identical) clusters consisting of a pair of antiferromagnetically coupled (J > 60 cm-1, H = JS1.S2) Fe3+ sites. The diferric cluster exhibited absorption bands from 300 to 355 nm; addition of azide elicited a charge transfer band at 450 nm. In the presence of dithionite, the clusters were reduced to the diferrous state. Addition of stearoyl-CoA and O2 returned the clusters to the diferric state. These properties are consistent with assigning the desaturase to the class of O2-activating proteins containing diiron-oxo clusters, most notably ribonucleotide reductase and methane monooxygenase hydroxylase. Comparison of the primary structures for these three catalytically diverse proteins revealed a conserved pair of the amino acid sequence -(Asp/Glu)-Glu-Xaa-Arg-His- separated by approximately 100 amino acids. Since each of these proteins can catalyze O2-dependent cleavage of unactivated C--H bonds, we propose that these amino acid sequences represent a biological motif used for the creation of reactive catalytic intermediates. Thus, eukaryotic fatty acid desaturation may proceed via enzymatic generation of a high-valent iron-oxo species derived from the diiron cluster.     

A model for the CO-inhibited form of [NiFe] hydrogenase: synthesis of (CO)3Fe(μ-StBu)3Ni{SC6H3-2,6-(mesityl)2} and reversible CO addition at the Ni site

A [NiFe] hydrogenase model compound having a distorted trigonal-pyramidal nickel center, (CO)₃Fe(μ-S^tBu)₃Ni(SDmp), 1 (Dmp = C₆H₃-2,6-(mesityl)₂), was synthesized from the reaction of the tetranuclear Fe-Ni-Ni-Fe complex [(CO)₃Fe(μ-S^tBu)₃Ni]₂(μ-Br)₂, 2 with NaSDmp at -40 °C. The nickel site of complex 1 was found to add CO or CN^tBu at -40 °C to give (CO)₃Fe(S^tBu)(μ-S^tBu)₂Ni(CO)(SDmp), 3, or (CO)₃Fe(S^tBu)(μ-S^tBu)₂Ni(CN^tBu)(SDmp), 4, respectively. One of the CO bands of 3, appearing at 2055 cm^-1 in the infrared spectrum, was assigned as the Ni-CO band, and this frequency is comparable to those observed for the CO-inhibited forms of [NiFe] hydrogenase. Like the CO-inhibited forms of [NiFe] hydrogenase, the coordination of CO at the nickel site of 1 is reversible, while the CN^tBu adduct 4 is more robust.     

Spectroscopic evidence for an engineered,catalytically active Trp radical that creates the unique reactivity of lignin peroxidase

The surface oxidation site (Trp-171) in lignin peroxidase (LiP) required for the reaction with veratryl alcohol a high-redox-potential (1.4 V) substrate, was engineered into Coprinus cinereus peroxidase (CiP) by introducing a Trp residue into a heme peroxidase that has similar protein fold but lacks this activity. To create the catalytic activity toward veratryl alcohol in CiP, it was necessary to reproduce the Trp site and its negatively charged microenvironment by means of a triple mutation. The resulting D179W+R258E+R272D variant was characterized by multifrequency EPR spectroscopy. The spectra unequivocally showed that a new Trp radical [g values of g_x = 2.0035(5), g_y = 2.0027(5), and g_z = 2.0022(1)] was formed after the [Fe(IV)=O Por^•+] intermediate, as a result of intramolecular electron transfer between Trp-179 and the porphyrin. Also, the EPR characterization crucially showed that [Fe(IV)=O Trp-179^•] was the reactive intermediate with veratryl alcohol. Accordingly, our work shows that it is necessary to take into account the physicochemical properties of the radical, fine-tuned by the microenvironment, as well as those of the preceding [Fe(IV)=O Por^•+] intermediate to engineer a catalytically competent Trp site for a given substrate. Manipulation of the microenvironment of the Trp-171 site in LiP allowed the detection by EPR spectroscopy of the Trp-171^•, for which direct evidence has been missing so far. Our work also highlights the role of Trp residues as tunable redox-active cofactors for enzyme catalysis in the context of peroxidases with a unique reactivity toward recalcitrant substrates that require oxidation potentials not realized at the heme site.     

Engineering a tRNA and aminoacyl-tRNA synthetase for the site-specific incorporation of unnatural amino acids into proteins in vivo

In an effort to expand the scope of protein mutagenesis, we have completed the first steps toward a general method to allow the site-specific incorporation of unnatural amino acids into proteins in vivo. Our approach involves the generation of an “orthogonal” suppressor tRNA that is uniquely acylated in Escherichia coli by an engineered aminoacyl-tRNA synthetase with the desired unnatural amino acid. To this end, eight mutations were introduced into tRNA₂^Gln based on an analysis of the x-ray crystal structure of the glutaminyl-tRNA aminoacyl synthetase (GlnRS)–tRNA₂^Gln complex and on previous biochemical data. The resulting tRNA satisfies the minimal requirements for the delivery of an unnatural amino acid: it is not acylated by any endogenous E. coli aminoacyl-tRNA synthetase including GlnRS, and it functions efficiently in protein translation. Repeated rounds of DNA shuffling and oligonucleotide-directed mutagenesis followed by genetic selection resulted in mutant GlnRS enzymes that efficiently acylate the engineered tRNA with glutamine in vitro. The mutant GlnRS and engineered tRNA also constitute a functional synthetase–tRNA pair in vivo. The nature of the GlnRS mutations, which occur both at the protein–tRNA interface and at sites further away, is discussed.