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Peroxidase Activity of a c-Type Cytochrome b5 in the Non-Native State is Comparable to that of Native Peroxidases

The design of artificial metalloenzymes has achieved tremendous progress, although few designs can achieve catalytic performances comparable to that of native enzymes. Moreover, the structure and function of artificial metalloenzymes in non-native states has rarely been explored. Herein, we found that a c-type cytochrome b5 (Cyt b5), N57C/S71C Cyt b5, with heme covalently attached to the protein matrix through two Cys?heme linkages, adopts a non-native state with an open heme site after guanidine hydrochloride (Gdn?HCl)-induced unfolding, which facilitates H2O2 activation and substrate binding. Stopped-flow kinetic studies further revealed that c-type Cyt b5 in the non-native state exhibited impressive peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase. This study presents an alternative approach to the design of functional artificial metalloenzymes by exploring enzymatic functions in non-native states.

Peroxidase Activity of a c-Type Cytochrome b5 in the Non-Native State is Comparable to that of Native Peroxidases

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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The single crystal X-ray structure of beta-hematin DMSO solvate grown in the presence of chloroquine, a beta-hematin growth-rate inhibitor

Single crystals of solvated beta-hematin were grown from a DMSO solution containing the antimalarial drug chloroquine, a known inhibitor of beta-hematin formation. In addition, a kinetics study employing biomimetic lipid-water emulsion conditions was undertaken to further investigate the effect of chloroquine and quinidine on the formation of beta-hematin. Scanning electron microscopy shows that the external morphology of the beta-hematin DMSO solvate crystals is almost indistinguishable from that of malaria pigment (hemozoin), and single crystal X-ray diffraction confirms the presence of mu-propionato coordination dimers of iron(III) protoporphyrin IX. The free propionic acid functional groups of adjacent dimers hydrogen bond to included DMSO molecules, rather than forming carboxylic acid dimers. The observed exponential kinetics were modeled using the Avrami equation, with an Avrami constant equal to 1. The decreased rate of beta-hematin formation observed at low concentrations of both drugs could be accounted for by assuming a mechanism of drug adsorption to sites on the fastest growing face of beta-hematin. This behavior was modeled using the Langmuir isotherm. Higher concentrations of drug resulted in decreased final yields of beta-hematin, and an irreversible drug-induced precipitation of iron(III) protoporphyrin IX was postulated to account for this. The model permits determination of the equilibrium adsorption constant (Kads). The values for chloroquine (log Kads = 5.55 ¡À 0.03) and quinidine (log Kads = 4.92 ¡À 0.01) suggest that the approach may be useful as a relative probe of the mechanism of action of novel antimalarial compounds.

The single crystal X-ray structure of beta-hematin DMSO solvate grown in the presence of chloroquine, a beta-hematin growth-rate inhibitor

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Elucidating connectivity and metal-binding structures of unlabeled paramagnetic complexes by13C and1H solid-state NMR under fast magic angle spinning

Characterizing paramagnetic complexes in solids is an essential step toward understanding their molecular functions. However, methodologies to characterize chemical and electronic structures of paramagnetic systems at the molecular level have been notably limited, particularly for noncrystalline solids. We present an approach to obtain connectivities of chemical groups and metal-binding structures for unlabeled paramagnetic complexes by 13C and 1H high-resolution solid-state NMR (SSNMR) using very fast magic angle spinning (VFMAS, spinning speed ?20 kHz). It is experimentally shown for unlabeled Cu(II)(Ala-Thr) that 2D 13CV1H correlation SSNMR under VFMAS provides the connectivity of chemical groups and assignments for the characterization of unlabeled paramagnetic systems in solids. We demonstrate that on the basis of the assignments provided by the VFMAS approach multiple13C-metal distances can be simultaneously elucidated by a combination of measurements of 13C anisotropic hyperfine shifts and 13C T1 relaxation due to hyperfine interactions for this peptide-Cu(II) complex. It is also shown that an analysis of 1H anisotropic hyperfine shifts allows for the determination of electron-spin states in Fe(III)-chloroprotoporphyin-IX in solid states.

Elucidating connectivity and metal-binding structures of unlabeled paramagnetic complexes by13C and1H solid-state NMR under fast magic angle spinning

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Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Self-assembly of hemin on carbon nanotube as highly active peroxidase mimetic and its application for biosensing

Hemin was assembled on the surface of single-walled carbon nanotubes (SWCNT) through non-covalent functionalization by pi-pi stacking. The resulting nanohybrid of hemin-SWCNT possessed an intrinsic peroxidase-like activity, and could effectively catalyze oxidation of the substrate 3,3,5,5-tetramethylbenzidine by H2O2 to develop a blue color in aqueous solution. The activity of hemin-SWCNT hybrid material was much higher than the activity of hemin alone. Combination of the catalytic reaction of glucose with glucose oxidase and the hemin-SWCNT hybrid catalytic reaction, a colorimetric method for glucose detection was also developed. The hemin-SWCNT nanohybrid also exhibited high stability and excellent reusability.

Self-assembly of hemin on carbon nanotube as highly active peroxidase mimetic and its application for biosensing

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Binding of azole drugs to heme: A combined MS/MS and computational approach

The binding properties of azole drugs toward ferric heme have been examined, focusing on well known antifungal drugs bearing imidazole and triazole heteroaromatic rings. These drugs are known to act as inhibitors of the Candida albicans P450 sterol 14alpha-demethylase enzyme, through binding to the heme prosthetic group. Absolute binding energies have been determined experimentally by energy variable collision induced dissociation experiments performed on the selected ionic complexes and evaluated theoretically using density functional theory, within the Car-Parrinello Molecular Dynamics method. The two series display some agreement in the relative binding energies data. These findings suggest that the combined ab initio and mass spectrometric approach may prove fruitful in assaying complexes between a prosthetic group and an array of ligands of potential pharmacological activity. It is shown that the axial interaction of the imidazole-based drugs with iron(III) is somewhat stronger than that of the triazole-based drugs. This general observation fails if specific interactions remote from the metal center come into play. For example, a hydrogen bond interaction is established in the ferric heme complex with fluconazole, a drug of the triazole family owning a hydroxyl group prone to interact with the carbonyl oxygen of a propionyl group on the periphery of protoporphyrin IX. However, the relatively uniform values for both the experimental and theoretically calculated binding energies underline the important role played by the prosthetic group environment in tuning the heme interaction with biological and xenobiotic molecules and ultimately in modulating enzyme activity.

Binding of azole drugs to heme: A combined MS/MS and computational approach

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity

The free form of human cytoplasmic arginyl-tRNA synthetase (hcArgRS) is hypothesized to participate in ubiquitin-dependent protein degradation by offering arginyl-tRNAArg to arginyl-tRNA transferase (ATE1). We investigated the effect of hemin on hcArgRS based on the fact that hemin regulates several critical proteins in the “N-end rule” protein degradation pathway. Extensive biochemical evidence has established that hemin could bind to both forms of hcArgRS in vitro. Based on the spectral changes of the Soret band on site-directed protein mutants, we identified Cys-115 as a specific axial ligand of hemin binding that is located in the Add1 domain. Hemin inhibited the catalytic activity of full-length and N-terminal 72-amino acid-truncated hcArgRSs by blocking amino acid activation. Kinetic analysis demonstrated that the Km values for tRNAArg, arginine, and ATP in the presence of hemin were not altered, but kcat values dramatically decreased compared with those in the absence of hemin. By comparison, the activity of prokaryotic ArgRS was not affected obviously by hemin. Gel filtration chromatography suggested that hemin induced oligomerization of both the isolated Add1 domain and the wild type enzyme, which could account for the inhibition of catalytic activity. However, the catalytic activity of an hcArgRS mutant with Cys-115 replaced by alanine (hcArgRS-C115A) was also inhibited by hemin, suggesting that hemin binding to Cys-115 is not responsible for the inhibition of enzymatic activity and that the specific binding may participate in other biological functions.

Hemin binds to human cytoplasmic arginyl-tRNA synthetase and inhibits its catalytic activity

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Dirhodium (II) complex interferes with iron-transport system to exert antibacterial action against Streptococcus pneumoniae

Drug resistance in bacteria is becoming a significant threat to global public health, and the development of novel and efficient antibacterial compounds is urgently needed. Recently, rhodium complexes have attracted attention as antimicrobial agents, yet their antibacterial mechanism remains unknown. In this study, we observed that the dirhodium (II) complex Rh2Ac4 inhibited Streptococcus. pneumoniae growth without significant cytotoxic side-effects on host cells in vitro. We subsequently investigated the antibacterial mechanism of Rh2Ac4 using iTRAQ-based proteomics combined with cellular and biochemical assays. Bioinformatics analysis on the proteomic alterations demonstrated that six molecular functional groups, including metal ion binding and twelve metabolic pathways, were significantly affected after treatment with Rh2Ac4. The interaction network analysis of metal ion binding proteins suggested that Rh2Ac4 decreased the protein expression levels of SPD_1652, SPD_1590 and Gap, which are associated with haem uptake/metabolism. Cellular and biochemical assays further confirmed that Rh2Ac4 could be taken up by bacteria via the PiuABCD haem-uptake system. The structurally similar Rh complex may compete with Fe-haem to decrease Fe-uptake via the PiuABCD system, disrupting iron metabolism to exert its antibacterial activity against S. pneumoniae. These data indicate that Rh2Ac4 is a promising new drug for the treatment of S. pneumoniae infections.

Dirhodium (II) complex interferes with iron-transport system to exert antibacterial action against Streptococcus pneumoniae

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Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Quinine and chloroquine differentially perturb heme monomer-dimer equilibrium

Nuclear magnetic resonance (NMR) measurements of magnetic susceptibility have been utilized to study the equilibrium between two forms (high-spin monomer vs the antiferromagnetically coupled mu-oxo dimer) of ferriprotoporphyrin(IX) as a function of pH. The pH dependence of this equilibrium is significantly altered by the addition of either chloroquine or quinine. Chloroquine promotes the mu-oxo dimer whereas quinine promotes the monomer.

Quinine and chloroquine differentially perturb heme monomer-dimer equilibrium

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration

Protein tyrosine nitration is implicated in the occurrence and progression of pathological conditions involving free radical reactions. It is well recognized that hemin can catalyze protein tyrosine nitration in the presence of nitrite and hydrogen peroxide. Generally, the catalytic efficiency is positively correlated to its peroxidase activity. In this study, however, it is found that the efficiency of hemin in catalyzing protein tyrosine nitration is largely suppressed after functionalization with graphene derivatives, even though its peroxidase-like activity is more than quadrupled. Further studies show that the oxidation of tyrosine is still observed for these composites; dityrosine formation, however, is greatly inhibited. Furthermore, these composites also exhibit strong effects on the oxidation of nitrite into nitrate. Therefore, we propose a mechanism in which hemin-graphene derivatives facilitate the oxidation of tyrosine and nitrite to produce tyrosyl radicals and nitrogen dioxide radicals in the presence of hydrogen peroxide, but graphene interlayers serve as barriers that hinder radical?radical coupling reactions; consequently, protein tyrosine nitration is restrained. This property of hemin-graphene derivatives, by which they catalyze substrate oxidation but suppress radical?radical coupling reactions, shows their great potential in selective oxidation procedures for byproduct removal.

Hemin-Graphene Derivatives with Increased Peroxidase Activities Restrain Protein Tyrosine Nitration

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion

 

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Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications

Rapid, high-throughput, and quantitative evaluations of biological metabolites in complex milieu are increasingly required for biochemical, toxicological, pharmacological, and environmental analyses. They are also essential for the development, testing, and improvement of new commercial chemical products. We demonstrate the application of ultra-high performance liquid chromatography-mass spectrometry (uHPLC-MS), employing an electrospray ionization source and a high accuracy quadrupole time-of-flight mass analyzer, for the identification and quantification of a series of porphyrin derivatives in liver: a matrix of particular relevance in toxicological or pharmacological testing. Exact mass is used to identify and quantify the metabolites. Chromatography enhances sensitivity and alleviates potential saturation issues by fanning out the contents of a complex sample before their injection into the spectrometer, but is not strictly necessary for the analysis. Extraction and sample treatment procedures are evaluated and matrix effects discussed. Using this method, the known mechanism of action of a well-characterized porphyrinogenic agent was verified in liver extracts from treated rats. The method was also validated for use with bacterial cells. This exact-mass method uses workhorse instruments available in many laboratories, providing a highly flexible alternative to existing HPLC- and MS/MS-based approaches for the simultaneous analysis of multiple compounds in biological media.

Mass-spectrometric profiling of porphyrins in complex biological samples with fundamental, toxicological, and pharmacological applications

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Reference£º
Iron Catalysis in Organic Synthesis | Chemical Reviews,
Iron Catalysis in Organic Synthesis: A Critical Assessment of What It Takes To Make This Base Metal a Multitasking Champion