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Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through hydrogen bonding interactions between proximal ligands and neighboring amino acids. Here, we show that introduction of a chemically programmed proximal Nd-methyl histidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His hydrogen bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher number of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic and kinetic characterization of APX2 NMH and several active site variants provides valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chemically optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins. [ABSTRACT FROM AUTHOR]

Two luminescent porous coordination polymers (PCPs), i.e., [Cu2(μ2-I)2ctpyz]n and [Cu4(μ3-I)4ctpyz]n (Cu2 and Cu4, respectively; ctpyz = cis-1,3,5-cyclohexanetriyl-2,2′,2″-tripyrazine), were successfully synthesized and characterized by single-crystal X-ray diffraction and luminescence spectroscopic measurements. Cu2 consists of rhombus-type dinuclear {Cu2I2} cores bridged by ctpyz ligands, while Cu4 is constructed of cubane-type tetranuclear {Cu4I4} cores bridged by ctpyz ligands. The void fraction of Cu4 is estimated to be 48.0%, which is significantly larger than that of Cu2 (19.9%). Under UV irradiation, both PCPs exhibit red luminescence at room temperature in the solid state (λem values of 660 and 614 nm for Cu2 and Cu4, respectively). Although the phosphorescence of Cu2 does not change upon removal and/or adsorption of EtOH solvent molecules in the porous channels, the solid-state emission maximum of Cu4 red-shifts by 36 nm (λem = 650 nm) upon the removal of the adsorbed benzonitrile (PhCN) molecules from the porous channels (and vice versa). This large difference in the vapochromic behavior of Cu2 and Cu4 is closely related to the framework flexibility. The framework of Cu2 is sufficiently rigid to retain the porous structure without solvated EtOH molecules, whereas the porous structure of Cu4 collapses easily after removal of the adsorbed PhCN molecules to form a nonporous amorphous phase. The original vapor-adsorbed porous structure of Cu4 is regenerated by exposure of the amorphous solid to not only PhCN vapor but also tetrahydrofuran, acetone, ethyl acetate, and N,N-dimethylformamide vapors. The Cu4 structures with the various adsorbed solvents showed almost the same emission maxima as the original PhCN-adsorbed Cu4, except for DMF-adsorbed Cu4, which showed no luminescence probably because of weak coordination of the DMF vapor molecules to the Cu(I) centers of the tetranuclear {Cu4I4} core. [ABSTRACT FROM AUTHOR]

A general technique for introducing biophysical probes into selected receptors in their native environment is valuable for the study of their structure, dynamics, function, and molecular interactions. A number of such techniques rely on genetic engineering, which is not applicable for the study of endogenous proteins, and such approaches often suffer from artifacts due to the overexpression and bulky size of the probes/protein tags used. Here we designed novel catalyst-antibody conjugates capable of introducing small chemical probes into receptor proteins such as epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in a selective manner on the surface of living cells. Because of the selectivity and efficiency of this labeling technique, we were able to monitor the cellular dynamics and lifetime of HER2 endogenously expressed on cancer cells. More significantly, the current labeling technique comprises a stable covalent bond, which combined with a peptide mass fingerprinting analysis allowed epitope mapping of antibodies on living cells and identification of potential binding sites of anti-EGFR affibody. Although as yet unreported in the literature, the binding sites predicted by our labeling method were consistently supported by the subsequent mutation and binding assay experiments. In addition, this covalent labeling method provided experimental evidence that HER2 exhibits a more dynamic structure than expected on the basis of crystallographic analysis alone. Our novel catalyst-antibody conjugates are expected to provide a general tool for investigating the protein trafficking, fluctuation, and molecular interactions of an important class of cell-surface receptors on live cell surfaces. [ABSTRACT FROM AUTHOR]

Two non-heme iron—nitrosyl species, [Fe2(N-Et-HPTB)(O2CPh)(NO) 2](BF4)2 (la ) and [Fe2(N-Et-HPTB) (DMF)2(NO)(OH)](BF4)3 (2a), are characterized by FTIR and resonance Raman spectroscopy. Binding of NO is reversible in both complexes, which are prone to NO photolysis under visible light illumination. Photoproduction of N2O occurs in high yield for la but not 2a. Low-temperature FTIR photolysis experiments with la in acetonitrile do not reveal any intermediate species, but in THF at room temperature, a new {FeNO}7 species quickly forms under illumination and exhibits a …NO ) vibration indicative of nitroxyl-like character. This metastable species reacts further under illumination to produce N2O. A reaction mechanism is proposed, and implications for NO reduction in flavodiiron proteins are discussed. [ABSTRACT FROM AUTHOR]

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/ copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N2O. Engineering a nonheme FeB site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N2O in solution and to show that the presence of a distal FeB II is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the dliron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five- coordinate low-spin {FeNO}7 species with v(FeNO)heme. and v(NO)heme at 522 and 1660 cm-1, and a second NO molecule is bound to the nonheme FeB site with a v(NO)FeB at 1755 cm-1. Stopped-flow UV-vis absorption coupled with rapid-freeze- quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to FeB is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex with characteristic z1(FeNO)heme at 570 ± 2 cm-1and v(NO)FeB at 1755 cm-1. Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in FeBMb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six- coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex to decay with production of N2O at a rate of 0.7 s-1 at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs. [ABSTRACT FROM AUTHOR]