Chromophore Formation Research Articles

Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications

Naturally occurring fluorescent proteins (FPs) cloned from marine organisms often suffer from many drawbacks for cell biology applications, including poor folding efficiency at 37 °C, slow chromophore formation and obligatory quaternary structure. Many of these drawbacks can be minimized or eliminated by using protein engineering and directed evolution, resulting in superior probes for use in live-cell fluorescence microscopy. In this protocol, we provide methods for engineering a monomeric FP, for enhancing its brightness by directed evolution, and for thoroughly characterizing the optimized variant. Variations on this procedure can be used to select for many other desirable features, such as a red-shifted emission spectrum or enhanced photostability. Although the length of the procedure is dependent on the degree of optimization desired, the basic steps can be accomplished in 4-6 weeks.

Dynamic light absorption of biomass-burning organic carbon photochemically aged under natural sunlight

Abstract. Wood-burning aerosol produced under smoldering conditions was photochemically aged with different relative humidity (RH) and NOx conditions using a 104 m3 dual outdoor chamber under natural sunlight. Light absorption of organic carbon (OC) was measured over the course of photooxidation using a UV–visible spectrometer connected to an integrating sphere. At high RH, the color decayed rapidly. NOx slightly prolonged the color of wood smoke, suggesting that NOx promotes the formation of chromophores via secondary processes. Overall, the mass absorption cross section (integrated between 280 and 600 nm) of OC increased by 11–54% (except high RH) in the morning and then gradually decreased by 19–68% in the afternoon. This dynamic change in light absorption of wood-burning OC can be explained by two mechanisms: chromophore formation and sunlight bleaching. To investigate the effect of chemical transformation on light absorption, wood smoke particles were characterized using various spectrometers. The intensity of fluorescence, which is mainly related to polycyclic aromatic hydrocarbons (PAHs), rapidly decreased with time, indicating the potential bleaching of PAHs. A decline of levoglucosan concentrations evinced the change of primary organic aerosol with time. The aerosol water content measured by Fourier transform infrared spectroscopy showed that wood-burning aerosol became less hygroscopic as photooxidation proceeded. A similar trend in light absorption changes has been observed in ambient smoke aerosol originating from the 2012 County Line wildfire in Florida. We conclude that the biomass-burning OC becomes less light absorbing after 8–9 h sunlight exposure compared to fresh wood-burning OC.

Protein-Chromophore Interactions in Green Fluorescent Protein (GFP) Studied by Split Protein Reconstitution

We have utilized a split green fluorescent protein (GFP) system to generate a range of semi-synthetic proteins to study the interaction of the autocatalytically-formed chromophore with the protein environment. Using circularly permuted GFP, the alpha helix containing the chromophore has been relocated to the N-terminus and then removed by site-specific proteolysis of an engineered loop region. Following denaturation and size exclusion chromatography purification, the truncated protein and the chromophore-bearing peptide are separated. We previously showed that the truncated protein is capable of re-forming a chromophore upon reconstitution with a synthetic peptide. This work is extended to incorporate unnatural amino acids into the chromophore for studying hydrogen bonding and proton transfer. An additional application we have pursued is to use chromophore "transplantation" in order to examine the spectroscopic effects of mutations that would ordinarily be impossible due to the preclusion of chromophore formation. Arg96, in particular, is an interesting residue because of its major role in defining the electrostatic environment, however, its intolerance to mutation necessitates the use of a split protein and complementation with a chromophore-containing peptide harvested from a non-mutated donor. We use this scheme to investigate the effects that remodeling the electrostatics at this critical position have on the color tuning and quantum yield of GFP.

Development ofβ-Lactamase as a Tool for Monitoring Conditional Gene Expression by a Tetracycline-Riboswitch inMethanosarcina acetivorans

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

Structural consequences of chromophore formation and exploration of conserved lid residues amongst naturally occurring fluorescent proteins

Computational methods were used to generate the lowest energy conformations of the immature precyclized forms of the 28 naturally occurring GFP-like proteins deposited in the pdb. In all 28 GFP-like proteins, the beta-barrel contracts upon chromophore formation and becomes more rigid. Our prior analysis of over 260 distinct naturally occurring GFP-like proteins revealed that most of the conserved residues are located in the top and bottom of the barrel in the turns between the β-sheets (Ong et al. 2011) [1]. Structural analyses, molecular dynamics simulations and the Anisotropic Network Model were used to explore the role of these conserved lid residues as possible folding nuclei. Our results are internally consistent and show that the conserved residues in the top and bottom lids undergo relatively less translational movement than other lid residues, and a number of these residues may play an important role as hinges or folding nuclei in the fluorescent proteins.

A NOVEL VISIBLE SPECTROPHOTOMETRIC METHOD FOR THE ESTIMATION OF BETAMETHASONE USING OXIDATION APPROACH

A simple, sensitive, rapid and accurate spectrophotometric method was developed and validated for the estimation of betamethasone in bulk and pharmaceutical dosage forms. The method was based on the formation of bluish green chromophore with 0.0 4 % of sodium di c hromate and concentrated sulphuric acid showing the absorption at 6 15 nm. The proposed method has permitted the quantification of betamethasone over linearity in the range of 5 - 30 µg/ mL . The method was validated as per ICH guidelines.

Photoyellowing of chemically modified chemithermomechanical pulps (CTMP) from Eucalyptus globulus under various atmospheres

Abstract Chemithermomechanical pulps (CTMP) were prepared from Eucalyptus globulus and photoirradiated, and the responses have been examined by means of ultraviolet (UV) microspectrophotometer. The absorbance in the UV spectra of fiber walls increased in the range of 300 of 350 nm as a result of photoirradiation, indicating the formation of conjugated double-bound systems in the fiber walls. These spectral changes occurred only on the irradiated surface of sheets. CTMP photoirradiation under various atmospheres indicated that the presence of oxygen in the irradiation atmosphere is not necessary for CTMP yellowing. In nitrogen or vacuum, photoyellowing was at the same level as observed under oxygen. This observation was interpreted as a result of direct elimination of proton radicals from free phenolic groups and/or cleavage of β-aryl ether linkages. This induced probably the excited triplet state of α-carbonyl structures in CTMP, which leads to chromophore formation in the absence of oxygen. Reduction of CTMP with sodium borohydride impeded effectively the effects of photoirradiation only in the absence of oxygen. On the contrary, acetylation of CTMP had no effect in the absence of oxygen but led to photobleaching in the presence of oxygen.

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λex/λem = 502/511 nm) and red laRFP (λex/λem ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C(β) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

Improving accuracy of cell and chromophore concentration measurements using optical density.

BackgroundUV–vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating chromophore formation and cell concentration in liquid culture. OD wavelength is often chosen with little thought given to its effect on the quality of the measurement. Analysis of the contributions of absorption and scattering to the measured optical density provides a basis for understanding variability among spectrophotometers and enables a quantitative evaluation of the applicability of the Beer-Lambert law. This provides a rational approach for improving the accuracy of OD measurements used as a proxy for direct dry weight (DW), cell count, and pigment levels.ResultsFor pigmented organisms, the choice of OD wavelength presents a tradeoff between the robustness and the sensitivity of the measurement. The OD at a robust wavelength is primarily the result of light scattering and does not vary with culture conditions; whereas, the OD at a sensitive wavelength is additionally dependent on light absorption by the organism’s pigments. Suitably robust and sensitive wavelengths are identified for a wide range of organisms by comparing their spectra to the true absorption spectra of dyes. The relative scattering contribution can be reduced either by measurement at higher OD, or by the addition of bovine serum albumin. Reduction of scattering or correlation with off-peak light attenuation provides for more accurate assessment of chromophore levels within cells. Conversion factors between DW, OD, and colony-forming unit density are tabulated for 17 diverse organisms to illustrate the scope of variability of these correlations. Finally, an inexpensive short pathlength LED-based flow cell is demonstrated for the online monitoring of growth in a bioreactor at culture concentrations greater than 5 grams dry weight per liter which would otherwise require off-line dilutions to obtain non-saturated OD measurements.ConclusionsOD is most accurate as a time-saving proxy measurement for biomass concentration when light attenuation is dominated by scattering. However, the applicability of OD-based correlations is highly dependent on the measurement specifications (spectrophotometer model and wavelength) and culture conditions (media type; growth stage; culture stress; cell/colony geometry; presence and concentration of secreted compounds). These variations highlight the importance of treating literature conversion factors as rough approximations as opposed to concrete constants. There is an opportunity to optimize measurements of cell pigment levels by considering scattering and absorption-dependent wavelengths of the OD spectrum.

Estimation of plasma triglycerides with correction for free glycerol by orlistat inhibition of lipoprotein lipase activity

This report presents a modification of the enzymatic method (lipoprotein lipase/glycerol kinase/glycerol-3-phosphate oxidase/peroxidase) for determination of plasma triglyceride levels in order to achieve correction for the free glycerol content. The strategy is based on elimination of lipoprotein lipase activity from the single “multienzyme reagent” by use of orlistat, thereby allowing formation of quinoneimine chromophore from free glycerol. Orlistat was found to abolish the lipoprotein lipase activity (triolein was used as substrate) without any impact on the glycerol-driven rate of quinoneimine generation. Conditions were standardized for estimation of both triglyceride and glycerol content of mice plasma using a microplate reader.

Elimination of Redundant and Stop Codons during the Chemical Synthesis of Degenerate Oligonucleotides. Combinatorial Testing on the Chromophore Region of the Red Fluorescent Protein mKate

Although some strategies have been reported for the elimination of stop and redundant codons during the chemical synthesis of degenerate oligonucleotides, incorporating an expensive cocktail of 20 trimer-phosphoramidites is currently a commonly employed and straightforward approach. As an alternative option, we describe here a cheaper strategy based on standard monomer-phosphoramidites and a simplified resin-splitting procedure. The accurate division of the resin, containing the growing oligonucleotide, into four columns represents the key step in this approach. The synthesis of the degenerate codon NDT in column 1, loaded with 60% of the resin, produces 12 codons, while a degenerate codon VMA in column 2, loaded with 30% of the resin, produces 6 codons. Codons ATG and TGG, independently synthesized in columns 3 and 4, respectively, and loaded with 5% each, completes the 20 different codons. The experimental frequency of each mutant codon in the library was assessed by randomizing 12 contiguous codons that encode for amino acids located in the chromophore region of the enhanced red fluorescent protein mKate-S158A. Furthermore, randomization of three contiguous codons that encode for the amino acids Phe62, Met63, and Tyr64, which are equivalent to Phe64, Ser65, and Tyr66 in GFP, gave rise to some red and golden yellow fluorescent mutants displaying interesting phenotypes and spectroscopic properties. The absorption and emission spectra of two of these mutants also suggested that the complete maturation of the red and golden yellow chromophores in mKate proceeds via the formation of a green-type chromophore and a cyan-type chromophore, respectively.

Brown carbon formation from ketoaldehydes of biogenic monoterpenes

Sources and chemical composition of brown carbon are poorly understood, and even less is known about the mechanisms of its atmospheric transformations. This work presents molecular-level investigations of the reactive compound ketolimononaldehyde (KLA, C9H14O3), a second-generation ozonolysis product of limonene (C10H16), as a potent brown carbon precursor in secondary organic aerosol (SOA) through its reactions with reduced nitrogen compounds, such as ammonium ion (NH4+), ammonia, and amino acids. The reactions of synthesized and purified KLA with NH4+ and glycine resulted in the formation of chromophores nearly identical in spectral properties and formation rates to those found in similarly-aged limonene/O3 SOA. Similar chemical reaction processes of limononaldehyde (LA, C10H16O2) and pinonaldehyde (PA, C10H16O2), the first-generation ozonolysis products of limonene and alpha-pinene, respectively, were also studied, but the resulting products did not exhibit the light absorption properties of brown carbon, suggesting that the unique molecular structure of KLA produces visible-light-absorbing compounds. The KLA/NH4+ and KLA/GLY reactions produce water-soluble, hydrolysis-resilient chromophores with high mass absorption coefficients (MAC = 2000-4000 cm2 g(-1)) at lambda - 500 nm, precisely at the maximum of the solar emission spectrum. Liquid chromatography was used to isolate the light-absorbing fraction, and UV-Vis, FTIR, NMR and high-resolution mass spectrometry (HR-MS) techniques were used to investigate the structures and chemical properties of the light-absorbing compounds. The KLA browning reaction generates a diverse mixture of light-absorbing compounds, with the majority of the observable products containing 1-4 units of KLA and 0-2 nitrogen atoms. Based on the HR-MS product distribution, conjugated aldol condensates, secondary imines (Schiff bases), and N-heterocycles like pyrroles may contribute in varying degree to the light-absorbing properties of the KLA brown carbon. The results of this study demonstrate the high degree of selectivity of organic compound structures on the light-absorbing properties of SOA.

A Compact Miniaturized Flow System Based on Low-Temperature Co-fired Ceramic Technology Coupled to LED Mini-photometer for Determination of Dipyrone in Pharmaceutical Formulations

In this work, an analytical microsystem based on LTCC (low-temperature co-fired ceramic) technology with monolithic incorporation of an optical flow cell for determination of dipyrone in pharmaceuticals is described. The detection system is based on the formation of a blue chromophore between dipyrone and Fe(III) photometrically monitored at 630 nm using a lab-made LED mini-photometer constructed with a light emitting diode as a radiation source and a Si photodiode as a detector. The lab-made mini-photometer elaborated presented a good performance in regard to high signal/noise ratio, low drift and good sensitivity. The analytical curve was linear in the dipyrone concentration range from 1.0 × 10 -4

In vitro and in vivo activity of a new unsymmetrical dinuclear copper complex containing a derivative ligand of 1,4,7-triazacyclononane: catalytic promiscuity of [Cu2(L)Cl3

Here we present the synthesis of the dinuclear complex [Cu(II)2(L)Cl3] (1), where L is the deprotonated form of the 3-[(4,7-diisopropyl-1,4,7-triazacyclononan-1-yl)methyl]-2-hydroxy-5-methylbenzaldehyde ligand. The complex was characterized by single crystal X-ray diffraction, potentiometric titration, mass spectrometry, electrochemical and magnetic measurements, EPR, UV-Vis and IR. Complex 1 is able to increase the hydrolysis rate of the diester bis-(2,4-dinitrophenyl)phosphate (2,4-BDNPP) by a factor of 2700, and also to promote the plasmidial DNA cleavage at pH 6 and to inhibit the formazan chromophore formation in redox processes at pH 7. Using Saccharomyces cerevisiae (BY4741) as a eukaryotic cellular model, we observed that 1 presents reduced cytotoxicity. In addition, treatment of wild-type and mutant cells lacking Cu/Zn-superoxide dismutase (Sod1) and cytoplasmic catalase (Ctt1) with 1 promotes increased survival after H2O2 or menadione (O2˙(-) generator) stress, indicating that 1 might act as a Sod1 and Ctt1 mimetic. Considered together, these results support considerations regarding the dynamic behaviour of an unsymmetrical dinuclear copper(II) complex in solid state and in aqueous pH-dependent solution.

Facial visible spectrophotometric determination of metformin hydrochloride in glucosam tablets and industrial waste water: Application to content uniformity testing

Objectives: To determine simple, accurate and highly sensitive spectrophotometric method for determination metformin hydrochloride in pharmaceutical preparations and in industrial waste water sample. Materials and Method: The method is based on the oxidation of metformin by a known excess of sodium hypochlorite in alkaline medium. Results: Formation of a yellow-colored chromophore having maximum absorbance at 385nm. Molar absorptivity was found to be 2 × 10 4 L.mol -1 .cm -1 . Beer’s law is obeyed in the concentration range of 0.5-4 µg .ml -1 , relative standard deviation (RSD) is better than ±1.8 (n =10) .The limits of detection and quantitation are 0.083 and 0.25 µg .mL -1 , respectively. The method is applied successfully to determination of metformin pharmaceutical formulation (tablets).The common excipients do not interfere with the proposed method. A statistical comparison of these results with those of official method using t and F values at 95% confidence level shows good agreements and indicated no significant difference in the precision, and the present method has good validity. Conclusion: The proposed method can be used as a routine quality control and content uniformity tests for determination of metformin in pure form, tablet formulations and industrial waste water sample.

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore.

Fluorescence and photodegradation of Xuan paper: The photostability of traditional Chinese handmade paper

Xuan paper is a type of Chinese handmade paper produced for traditional calligraphy and painting in China since the Tang Dynasty (618–907 AD), and is therefore an important cultural heritage material. In this study, the fluorescence and photochemical properties of Xuan paper were investigated. Xuan paper exhibits auto-fluorescence in the blue spectral range (450–500 nm) and UVA photolysis of the paper resulted in a substantial reduction in the blue fluorescence together with the formation of chromophores absorbing in the visible spectrum, resulting in photoyellowing of the paper. A more significant yellowing of paper was observed when irradiated in the dry state than under wet conditions. The associated photogeneration of hydrogen peroxide and superoxide from three types of Xuan paper samples during irradiation showed a correlation between the yields of reactive oxygen species and their relative yellowing rates. The results are interpreted in terms of sensitised photooxidation via a mechanism of electron transfer involving the fluorophores in their excited singlet states being responsible for the photodegradation of Xuan paper. SEM/EDS analyses were performed on the Xuan paper samples to investigate their morphological and elemental characteristics. Silica-containing fibres characteristic of a special rice straw that grows in siliceous soil were observed in all types of Xuan paper. Micron-sized calcium precipitates possibly formed from the “lime-steaming” manufacture process were shown to protect against the acidification of paper during accelerated thermal ageing.

Synthesis and Postfunctionalization of Rod–Coil Diblock and Coil–Rod–Coil Triblock Copolymers Composed of Poly(3-hexylthiophene) and Poly(4-(4′-N,N-dihexylaminophenylethynyl)styrene) Segments

Poly(3-hexylthiophene) (P3HT) with a bromobutyl functional group at the ω-chain-end (P3HT-C4Br) and P3HT with bromobutyl functional groups at the α,ω-chain-ends (BrC4-P3HT-C4Br) were synthesized by selecting the appropriate initiators for the Grignard metathesis (GRIM) polymerization. The high end-functionality was confirmed by matrix assisted laser desorption-ionization time-of-flight (MALDI–TOF) mass spectrometry. These polymers were efficiently reacted with the living anionic polymers of 4-(4′-N,N-dihexylaminophenylethynyl)styrene (DHPS) to yield novel rod–coil diblock and coil–rod–coil triblock copolymers composed of rigid P3HT and flexible poly(4-(4′-N,N-dihexylaminophenylethynyl)styrene) (PDHPS) segments. The expected structures of the block copolymers were confirmed by size exclusion chromatography (SEC), proton nuclear magnetic resonance (1H NMR), and Fourier transform infrared (FT-IR) spectroscopies. Furthermore, the side chain alkynes of the PDHPS segments of both P3HT-b-PDHPS and PDHPS-b-P3HT-b-PDHPS were quantitatively functionalized by a [2 + 2] cycloaddition followed by a cycloreversion with tetracyanoethylene (TCNE), producing the corresponding block copolymers with donor–acceptor moieties in the flexible polystyrene segments. The formation of the new chromophores was confirmed by UV–vis spectroscopy and cyclic voltammetry (CV), which revealed strong intramolecular charge-transfer bands and redox activities ascribed to the formed donor–acceptor moieties. The thermal properties and surface morphology of the block copolymers were also evaluated by differential scanning calorimetry (DSC), atomic force microscopy (AFM) observations, and small-angle and wide-angle X-ray scattering (SAXS and WAXS). This is the first report about the development of P3HT-based block copolymers with tunable optoelectronic properties, which was achieved by the combined synthetic techniques of the GRIM polymerization, living anionic polymerization, and click postfunctionalization.

Red fluorescent proteins: chromophore formation and cellular applications

In the last decade, a number of red fluorescent proteins (RFPs) that emit orange, red, and far-red fluorescence have been isolated from anthozoans (corals), and developed through directed molecular evolution. An attractive property possessed by some RFPs is that their red fluorescence can be turned on or modulated by illumination at specific wavelengths. Recent progress in the development of RFPs has been accompanied with detailed studies of chromophore chemistry. A thorough understanding of the molecular mechanisms involved in the post-translational modifications of red chromophores would enable scientists to design RFPs with the desired properties to advance imaging applications. This article provides a broad perspective on the chemistry and applications of RFPs.

Fe(II) complexes that mimic the active site structure of acetylacetone dioxygenase: O2 and NO reactivity.

Acetylacetone dioxygenase (Dke1) is a bacterial enzyme that catalyzes the dioxygen-dependent degradation of β-dicarbonyl compounds. The Dke1 active site contains a nonheme monoiron(II) center facially ligated by three histidine residues (the 3His triad); coordination of the substrate in a bidentate manner provides a five-coordinate site for O(2) binding. Recently, we published the synthesis and characterization of a series of ferrous β-diketonato complexes that faithfully mimic the enzyme-substrate intermediate of Dke1 (Park, H.; Baus, J.S.; Lindeman, S.V.; Fiedler, A.T. Inorg. Chem.2011, 50, 11978-11989). The 3His triad was modeled with three different facially coordinating N3 supporting ligands, and substituted β-diketonates (acac(X)) with varying steric and electronic properties were employed. Here, we describe the reactivity of our Dke1 models toward O(2) and its surrogate nitric oxide (NO), and report the synthesis of three new Fe(II) complexes featuring the anions of dialkyl malonates. Exposure of [Fe((Me2)Tp)(acac(X))] complexes (where (R2)Tp = hydrotris(pyrazol-1-yl)borate with R-groups at the 3- and 5-positions of the pyrazole rings) to O(2) at -70 °C in toluene results in irreversible formation of green chromophores (λ(max) ∼750 nm) that decay at temperatures above -60 °C. Spectroscopic and computational analyses suggest that these intermediates contain a diiron(III) unit bridged by a trans μ-1,2-peroxo ligand. The green chromophore is not observed with analogous complexes featuring (Ph2)Tp and (Ph)TIP ligands (where (Ph)TIP = tris(2-phenylimidazoly-4-yl)phosphine), since the steric bulk of the phenyl substituents prevents formation of dinuclear species. While these complexes are largely inert toward O(2), (Ph2)Tp-based complexes with dialkyl malonate anions exhibit dioxygenase activity and thus serve as functional Dke1 models. The Fe/acac(X) complexes all react readily with NO to yield high-spin (S = 3/2) {FeNO}(7) adducts that were characterized with crystallographic, spectroscopic, and computational methods. Collectively, the results presented here enhance our understanding of the chemical factors involved in the oxidation of aliphatic substrates by nonheme iron dioxygenases.