Spectroscopic studies of 1-aminocyclopropane-1-carboxylic acid oxidase: molecular mechanism and CO(2) activation in the biosynthesis of ethylene

1-Aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACCO) catalyzes the last step in the biosynthesis of the gaseous plant hormone ethylene, which is involved in development, including germination, fruit ripening, and senescence. ACCO is a mononuclear non-heme ferrous enzyme that couples the oxidation of the cosubstrate ascorbate to the oxidation of substrate ACC by dioxygen. In addition to substrate and cosubstrate, ACCO requires the activator CO(2) for continuous turnover. NIR circular dichroism and magnetic circular dichroism spectroscopies have been used to probe the geometric and electronic structure of the ferrous active site in ACCO to obtain molecular-level insight into its catalytic mechanism. Resting ACCO/Fe(II) is coordinatively saturated (six-coordinate). In the presence of CO(2), one ferrous ligand is displaced to yield a five-coordinate site only when both the substrate ACC and cosubstrate ascorbate are bound to the enzyme. The open coordination position allows rapid O(2) activation for the oxidation of both substrates. In the absence of CO(2), ACC binding alone converts the site to five-coordinate, which would react with O(2) in the absence of ascorbate and quickly deactivate the enzyme. These studies show that ACCO employs a general strategy similar to other non-heme iron enzymes in terms of opening iron coordination sites at the appropriate time in the reaction cycle and define the role of CO(2) as stabilizing the six-coordinate ACCO/Fe(II)/ACC complex, thus preventing the uncoupled reaction that inactivates the enzyme.     

ENDOR studies of the ligation and structure of the non-heme iron site in ACC oxidase

Ethylene is a plant hormone involved in all stages of growth and development, including regulation of germination, responses to environmental stress, and fruit ripening. The final step in ethylene biosynthesis, oxidation of 1-aminocyclopropane-1-carboxylic acid (ACC) to yield ethylene, is catalyzed by ACC oxidase (ACCO). In a previous EPR and ENDOR study of the EPR-active Fe(II)-nitrosyl, [FeNO],(7) complex of ACCO, we demonstrated that both the amino and the carboxyl moieties of the inhibitor d,l-alanine, and the substrate ACC by analogy, coordinate to the Fe(II) ion in the Fe(II)-NO-ACC ternary complex. In this report, we use 35 GHz pulsed and CW ENDOR spectroscopy to examine the coordination of Fe by ACCO in more detail. ENDOR data for selectively (15)N-labeled derivatives of substrate-free ACCO-NO (E-NO) and substrate/inhibitor-bound ACCO-NO (E-NO-S) have identified two histidines as protein-derived ligands to Fe; (1,2)H and (17)O ENDOR of samples in D(2)O and H(2)(17)O solvent have confirmed the presence of water in the substrate-free Fe(II) coordination sphere (E-NO). Analysis of orientation-selective (14,15)N and (17)O ENDOR data is interpreted in terms of a structural model of the ACCO active site, both in the presence (E-NO-S) and in the absence (E-NO) of substrate. Evidence is also given that substrate binding dictates the orientation of bound O(2).     

Crystal structure and mechanistic implications of 1-aminocyclopropane-1-carboxylic acid oxidase--the ethylene-forming enzyme

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). ACCO requires bicarbonate as an activator and catalyzes the oxidation of ACC to give ethylene, CO2, and HCN. We report crystal structures of ACCO in apo-form (2.1 A resolution) and complexed with Fe(II) (2.55 A) or Co(II) (2.4 A). The active site contains a single Fe(II) ligated by three residues (His177, Asp179, and His234), and it is relatively open compared to those of the 2-oxoglutarate oxygenases. The side chains of Arg175 and Arg244, proposed to be involved in binding bicarbonate, project away from the active site, but conformational changes may allow either or both to enter the active site. The structures will form a basis for future mechanistic and inhibition studies.     

In vitro germination of Striga hermonthica and Striga aspera seeds by 1-aminocyclopropane-1-carboxylic acid

Treatment of conditioned seeds of four isolates of Striga hermonthica and one isolate of Striga aspera with various concentrations of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), caused complex stimulation of germination patterns. GR 24, the strigol analogue served as a positive control and its stimulatory activity was comparable to that of ACC. When conditioned Striga seeds were treated with negative control that did not contain ACC, the stimulatory effect was lost. Overall, the germination data suggested a hormonal mode of action by ACC, which involves indirect stimulation of biosynthesis of ethylene that then triggers seed germination. The various mechanisms that have been proposed for the chemical and biological oxidation of ACC to generate ethylene are discussed.     

Synthesis of labeled 1-amino-2-methylenecyclopropane-1-carboxylic acid, an inactivator of 1-aminocyclopropane-1-carboxylate deaminase

1-Amino-2-methylenecyclopropane-1-carboxylic acid (2-methylene-ACC) is an irreversible inhibitor for a bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which catalyzes the conversion of ACC to alpha-ketobutyrate and ammonia. The inactivation has been proposed to proceed with the ring scission induced by an addition of an enzyme nucleophile, resulting in the formation of a reactive turnover product that then traps an active-site residue. To gain further insight into this unique enzymatic reaction, the tritiated 2-methylene-ACC was prepared and incubated with ACC deaminase to locate and identify the entrapped amino acid residue. The synthesis of this radiolabeled compound and the results of its incubation with ACC deaminase are reported in this paper.     

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.     

Extraction and quantitative analysis of 1-aminocyclopropane-1-carboxylic acid in plant tissue by gas chromatography coupled to mass spectrometry

We developed a new method for the determination of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) using quantitative GC-negative chemical ionisation MS as a detection and quantification system, in combination with isotope dilution using [2H4]ACC and an off-line solid-phase extraction. By derivatisation with pentafluorobenzyl bromide, ACC could easily be detected with m/z 280 being the most abundant ion. Determination of this component resulted in a detection limit of 10 fmol and a linear fit in the 100 fmol-100 pmol range. The combination of a rapid, high yield purification method with a stable derivatisation procedure and a sensitive detection method allowed the detection of ACC in samples as low as 100 mg fresh mass.     

Exploring the possibility of high-valent copper in models of copper proteins with a three-histidine copper-binding motif

An important function of many copper-containing proteins is activation of O2 and subsequent substrate oxidation. The Cu (III) oxidation state is generally considered to be less accessible because of the highly positive Cu (III)/Cu (II) redox potentials with typical amino acid ligands. Here, we employ density functional (DFT) calculations to explore to what extent copper (III) may be accessed in a biologically-relevant coordination environment around a mononuclear copper center, by breaking the oxygen-oxygen bond in a copper-(hydro) peroxide complex. In agreement with previous findings by Solomon and co-workers on copper models with related coordination patterns, the formally high-valent copper complex produced by O-O bond cleavage appears to harbor both oxidizing equivalents on the ligands. The potential energy surface for such a reaction reveals that with the three-histidine binding motif at the copper, O-O bond cleavage is not impossible, but rather disfavored thermodynamically.      

Multiple active intermediates in oxidation reaction catalyzed by synthetic heme-thiolate complex relevant to cytochrome p450

We have studied oxidation reactions using a synthetic heme-thiolate (SR complex) in order to ascertain the contributions of multiple intermediates derived from heme-thiolate to the oxygen atom transfer reaction to substrate. First, degradation of peroxyphenylacetic acid (PPAA) was examined in the presence of various substrates. The O-O bond cleavage mode of PPAA was clearly dependent on the reactivity of the substrate, and an easily oxidizable substrate enhanced heterolytic O-O bond cleavage. Second, competitive oxidations of cyclooctane and cyclooctene were carried out with various peroxybenzoic acids containing a series of substituents at the para-position as an oxygen source. The ratios of alkane hydroxylation rate/alkene epoxidation rate were dependent on the nature of the para-substituent of the oxidant. We conclude that substrate and oxidant interact with each other during the oxygen atom transfer reaction, that is, oxidation reaction occurs before O-O bond cleavage, even in the reaction catalyzed by heme-thiolate, which is considered to promote O-O bond cleavage. The results of an (18)O-incorporation study that is frequently performed to determine the active intermediates derived from iron porphyrins were consistent with this conclusion.     

A Novel Synthesis of 1-Aminocyclopropane-1-carboxylic Acid (ACC)

A novel synthetic route of 1-aminocyclopropane-1-carboxylic acid (ACC) has been built via cyclopropanedicarboxylic acid monohydrazide III in moderation to good yield.     

A hybrid density functional study of O-O bond cleavage and phenyl ring hydroxylation for a biomimetic non-heme iron complex

Density functional calculations using the B3LYP functional have been used to study the reaction mechanism of [Fe(Tp(Ph2))BF] (Tp(Ph2) = hydrotris(3,5-diphenylpyrazol-1-yl)borate; BF = benzoylformate) with dioxygen. This mononuclear non-heme iron(II) complex was recently synthesized, and it proved to be the first biomimetic complex reproducing the dioxygenase activity of alpha-ketoglutarate-dependent enzymes. Moreover, the enthalpy and entropy of activation for this biologically interesting process were derived from kinetic experiments offering a unique possibility for direct comparison of theoretical and experimental data. The results reported here support a mechanism in which oxidative decarboxylation of the keto acid is the rate-limiting step. This oxygen activation process proceeds on the septet potential energy surface through a transition state for a concerted O-O and C-C bond cleavage. In the next step, a high-valent iron-oxo species performs electrophilic attack on the phenyl ring of the Tp(Ph2) ligand leading to an iron(III)-radical sigma-complex. Subsequent proton-coupled electron-transfer yields an iron(II)-phenol intermediate, which can bind dioxygen and reduce it to a superoxide radical. Finally, the protonated superoxide radical leaves the first coordination sphere of the iron(III)-phenolate complex and dismutates to dioxygen and hydrogen peroxide. The calculated activation barrier (enthalpy and entropy) and the overall reaction energy profile agree well with experimental data. A comparison to the enzymatic process, which is suggested to occur on the quintet surface, has been made.     

Spectroscopic and kinetic studies of perturbed trinuclear copper clusters: the role of protons in reductive cleavage of the O-O bond in the multicopper oxidase Fet3p

The multicopper oxidase Fet3p couples four 1e(-) oxidations of substrate to the 4e(-) reduction of O2 to H2O. Fet3p uses four Cu atoms to accomplish this reaction: the type 1, type 2, and coupled binuclear type 3 sites. The type 2 and type 3 sites together form a trinuclear Cu cluster (TNC) which is the site of O2 reduction. This study focuses on mutants of two residues, E487 and D94, which lie in the second coordination sphere of the TNC and defines the role that each plays in the structural integrity of the TNC, its reactivity with O2, and in the directional movement of protons during reductive cleavage of the O-O bond. The E487D, E487A, and D94E mutants have been studied in the holo and type 1 depleted (T1D) forms. Residue E487, located near the T3 center, is found to be responsible for donation of a proton during the reductive cleavage of the O-O bond in the peroxide intermediate and an inverse kinetic solvent isotope effect, which indicates that this proton is already transferred when the O-O bond is cleaved. Residue D94, near the T2 site, plays a key role in the reaction of the reduced TNC with O2 and drives electron transfer from the T2 Cu to cleave the O-O bond by deprotonating the T2 Cu water ligand. A mechanism is developed where these second sphere residues participate in the proton assisted reductive cleavage of the O-O bond at the TNC.     

RhI‐Catalyzed Benzo/[7+1] Cycloaddition of Cyclopropyl‐Benzocyclobutenes and CO by Merging Thermal and Metal‐Catalyzed CC Bond Cleavages

A Rh‐catalyzed benzo/[7+1] cycloaddition of cyclopropyl‐benzocyclobutenes (CP‐BCBs) and CO to benzocyclooctenones has been developed. In this reaction, CP‐BCB acts as a benzo/7‐C synthon and the reaction involves two C?C bond cleavages: a thermal electrocyclic ring‐opening of the four‐membered ring in CP‐BCB and a Rh‐catalyzed C?C cleavage of the cyclopropane ring.     

Halonium-initiated C-O bond formation via umpolung of α-carbon to the carbonyl: efficient access to 5-amino-3(2H)-furanones

Highly efficient C-O bond formation has been developed via carboxylic acid catalyzed reaction of 1-acetylcyclopropanecarboxamides with N-halosuccinimide (NXS), which provides strategically novel and atom-economic access to biologically important 5-amino-3(2H)-furanones. The mechanism of halonium-initiated tandem oxa-cyclization and ring opening of cyclopropane was proposed. A variety of nucleophiles were found to open the cyclopropane.     

In silico structural and functional analysis of Mesorhizobium ACC deaminase

Nodulation is one of the very important processes of legume plants as it is the initiating event of fixing nitrogen. Although ethylene has essential role in normal plant metabolism but it has also negative impact on plants particularly in nodule formation in legume plants. It is also produced due to a variety of biotic or abiotic stresses. 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase is a rhizobial enzyme which cleaves ACC (immediate precursor of ethylene) into α-ketobutyrate and ammonia. As a result, the level of ethylene from the plant cells is decreased and the negative impact of ethylene on nodule formation is reduced. ACC deaminase is widely studied in several plant growth promoting rhizobacterial (PGPR) strains including many legume nodulating bacteria like Mesorhizobium sp. It is an important symbiotic nitrogen fixer belonging to the class – alphaproteobacteria under the order Rhizobiales. ACC deaminase has positive role in Legume-rhizobium symbiosis. Rhizobial ACC deaminase has the potentiality to reduce the adverse effects of ethylene, thereby triggering the nodulation process. The present study describes an in silico comparative structural (secondary structure prediction, homology modeling) and functional analysis of ACC deaminase from Mesorhizobium spp. to explore physico-chemical properties using a number of bio-computational tools. M. loti was selected as a representative species of Mesorhizobium genera for 3D modelling of ACC deaminase protein. Correlation by the phylogenetic relatedness on the basis of both ACC deaminase enzymes and respective acdS genes of different strains of Mesorhizobium has also studied.     

Catalytic reaction mechanism of homogentisate dioxygenase: a hybrid DFT study

Human homogentisate dioxygenase is an Fe(II)-dependent enzyme responsible for aromatic ring cleavage. The mechanism of its catalytic reaction has been investigated with the hybrid density functional method B3LYP. A relatively big model of the active site was first used to determine the substrate binding mode. It was found that binding of the substrate dianion with a vacant position trans to Glu341 is most favorable. The model was then truncated to include only the most relevant parts of the active-site residues involved in iron coordination and substrate binding. Thus, methylimidazole was used to model His292, His335, His365, and His371, while propionate modeled Glu341. The computational results suggest that the catalytic reaction of homogentisate dioxygenases involves three major chemical steps: formation of the peroxo intermediate, homolytic cleavage of the O-O bond leading to an arene oxide radical, and finally, cleavage of the six-membered ring. Calculated barriers for alternative reaction paths are markedly higher than for the proposed mechanism, and thus the computational results successfully explain the product specificity of the enzyme. Interestingly, the results indicate that the type of ring scission, intra or extra with respect to the substituents coordinating to iron, is controlled by the barrier heights for the decay of the arene oxide radical intermediate.     

Determination of 1-aminocyclopropane-1-carboxylic acid in apple extracts by capillary electrophoresis with laser-induced fluorescence detection

A rapid and sensitive method for the determination of 1-aminocyclopropane-1-carboxylic acid (ACC) in apple tissues has been described. This method is based on the derivatization of ACC with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ), and separation and quantification of the resulting FQ-ACC derivative by capillary electrophoresis coupled to laser-induced fluorescence detection (CE-LIF). Our results indicated that ACC derivatized with FQ could be well separated from other interfering amino acids using 20 mM borate buffer (pH 9.35) containing 40 mM sodium dodecyl sulfate and 10 mM Brij 35. The linearity of ACC was determined in the range from 0.05 to 5 microM with a correlation of 0.9967. The concentration detection limit for ACC was 10 nM (signal-to-noise = 3). The sensitivity and selectivity of this described method allows the analysis of ACC in crude apple extracts without extra purification and enrichment procedure.     

Ring opening reactions of pyromellitic dianhydride for the synthesis of first row transition metal dicarboxylate complexes

The ring opening reaction of pyromellitic dianhydride by methanol is an effective method to prepare first row transition metal dicarboxylate complexes. The reactions of different first row transition metal salts with pyromellitic dianhydride in the presence of nitrogen donating bidentate ligands such as 1,10-phenanthroline and 2,2′-bipyridine gives different compositions depending on the ligand and the metal salts used. For example, the reaction of nickel(II) acetate with pyromellitic dianhydride in the presence of 1,10-phenanthroline results in the formation of a carboxylato bridged nickel(II) metallacycle through the ring opening reaction of pyromellitic dianhydride (PAH) at the 1 and 3-positions, whereas a mononuclear tetra-aqua 2,2′-bipyridine nickel(II) complex is formed in a similar reaction of nickel(II) acetate through ring opening at the 1,4-position of PAH. Mononuclear cobalt(II) dicarboxylate complexes are formed from the ring opening reaction of pyromellitic dianhydride in methanol in the presence of the nitrogen donor ligands 1,10-phenanthroline or 2,2′-bipyridine. Copper(II) chloride on reaction with PAH and 2,2′-bipyridine gives a mononuclear complex via ring opening at the 1 and 4-positions; having chlorides inside and outside the coordination sphere. Whereas, the reaction of copper(II)acetate gives dinuclear copper complexes having a monodentate carboxylato bridge arising from the carboxylato groups at the 1 and 4-positions on the aromatic ring. The crystal structures of all the complexes have been determined.     

A density functional study of O-O bond cleavage for a biomimetic non-heme iron complex demonstrating an Fe(v)-intermediate

Density functional theory using the B3LYP hybrid functional has been employed to investigate the reactivity of Fe(TPA) complexes (TPA = tris(2-pyridylmethyl)amine), which are known to catalyze stereospecific hydrocarbon oxidation when H(2)O(2) is used as oxidant. The reaction pathway leading to O-O bond heterolysis in the active catalytic species Fe(III)(TPA)-OOH has been explored, and it is shown that a high-valent iron-oxo intermediate is formed, where an Fe(V) oxidation state is attained, in agreement with previous suggestions based on experiments. In contrast to the analogous intermediate [(Por.)Fe(IV)=O](+1) in P450, the TPA ligand is not oxidized, and the electrons are extracted almost exclusively from the mononuclear iron center. The corresponding homolytic O-O bond cleavage, yielding the two oxidants Fe(IV)=O and the OH. radical, has also been considered, and it is shown that this pathway is inaccessible in the hydrocarbon oxidation reaction with Fe(TPA) and hydrogen peroxide. Investigations have also been performed for the O-O cleavage in the Fe(III)(TPA)-alkylperoxide species. In this case, the barrier for O-O homolysis is found to be slightly lower, leading to loss of stereospecificity and supporting the experimental conclusion that this is the preferred pathway for alkylperoxide oxidants. The difference between hydroperoxide and alkylperoxide as oxidant derives from the higher O-O bond strength for hydrogen peroxide (by 8.0 kcal/mol).     

Synthesis and some transformations of bicyclic γ-lactones with a fused cyclopropane ring

Reductive cyclization of 4-hydroxymethyl-5,5-dimethyl(or pentamethylene)-2,5-dihydrofuran-2-ones by the action of sodium tetrahydridoborate gave bicyclic compounds in which the lactone ring is fused to a cyclopropane ring. Hydrolysis of the products with aqueous sodium hydroxide resulted in the formation of the corresponding disodium cyclopropane-1,1-dicarboxylates, which reacted with alkyl halides to produce the diesters. Acid hydrolysis of the fused systems was accompanied by opening of the cyclopropane ring with formation of 4-chloromethyl-5,5-dimethyl-2-oxotetrahydrofuran-3-carboxylic acid.