Ferrous Iron Binding Key to Mms6 Magnetite Biomineralisation: A Mechanistic Study to Understand Magnetite Formation Using pH Titration and NMR Spectroscopy

Formation of magnetite nanocrystals by magnetotactic bacteria is controlled by specific proteins which regulate the particles’ nucleation and growth. One such protein is Mms6. This small, amphiphilic protein can self‐assemble and bind ferric ions to aid in magnetite formation. To understand the role of Mms6 during in vitro iron oxide precipitation we have performed in situ pH titrations. We find Mms6 has little effect during ferric salt precipitation, but exerts greatest influence during the incorporation of ferrous ions and conversion of this salt to mixed‐valence iron minerals, suggesting Mms6 has a hitherto unrecorded ferrous iron interacting property which promotes the formation of magnetite in ferrous‐rich solutions. We show ferrous binding to the DEEVE motif within the C‐terminal region of Mms6 by NMR spectroscopy, and model these binding events using molecular simulations. We conclude that Mms6 functions as a magnetite nucleating protein under conditions where ferrous ions predominate.     

Study of the oxidative half‐reaction catalyzed by a non‐heme ferrous catalytic center by means of structural and computational methodologies

Deacetoxycephalosporin C synthase (DAOCS) is a mononuclear ferrous enzyme that catalyzes the expansion of the five‐membered thiazolidine ring of the penicillin nucleus into the six‐membered dihydrothiazine ring of the cephalosporins. In the first half‐reaction with dioxygen and 2‐oxoglutarate, a reactive iron–oxygen species is produced that can subsequently react with the penicillin substrate to yield the cephalosporin. We describe quantum mechanical calculations of the first part of the reaction based on the high‐resolution structures of the active site of DAOCS and its complexes with ligands. These studies are aimed at understanding how the reactive species can be produced and contained in the active site of the enzyme. The results demonstrate the priming of the active site by the co‐substrate for oxygen binding and hint to the presence of a stable iron–peroxo intermediate in equilibrium with a more reactive ferryl species and the formation of CO₂ as a leaving group by decarboxylation of 2‐oxoglutarate. A conclusion from these studies is that substitution of CO₂ by the penicillin substrate triggers the oxidation reaction in a booby‐trap‐like mechanism. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N¹‐methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate‐determining hydrogen‐atom abstraction on competitive σ‐ and π‐pathways on a quintet spin‐state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen‐bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.     

PHOTO-INDUCED BINDING OF SULFANILAMIDE TO CELLULAR MACROMOLECULES

Abstract— Ultraviolet light (A > 295 nm) induced binding of sulfanilamide to cellular macromoleculcs has been examined. It was found that the drug bound irreversibly to native DNA, and complexes containing one drug molecule per 80 nucleotides were obtained after 60 min of irradiation under anaerobic conditions. Oxygen reduced this binding significantly. More drug was bound to RNA and heat denatured DNA under identical conditions. The binding of sulfanilamide to DNA was found to induce nicking of circular closed plasmid DNA and cross-linking of calf thymus DNA. Oxygen significantly decreased nicking and cross-linking of DNA. Irradiation of sulfanilamide and human serum albumin resulted in covalent binding of the drug to the protein and a concomitant increase in protein crosslink-ing. While oxygen decreased covalent binding, crosslinking increased under aerobic conditions. These reactions may be important in the photosensitization caused by sulfanilamide.     

Analysis of the iron state in ferric and ferrous iron containing pharmaceutical products by Mössbauer spectroscopy

Summary An analysis of the iron state in commercial pharmaceuticals containing ferric and ferrous iron compounds, which are used for treatment of iron deficiency, was made by M?ssbauer spectroscopy. Small variations of the FeOOH cores of injectable iron-dextran complexes were observed. The presence of ferrous impurity in iron-dextran complexes was found. Characterization of the iron state in vitamins and dietary supplements containing ferrous compounds was made. The presence of ferrous and ferric impurities and iron compounds that were in disagreement with compounds announced by the manufacturer was detected by M?ssbauer spectroscopy.     

三种铁制剂治疗缺铁性贫血的疗效比较

在妇女、孕妇中,有些由于失血而失铁或孕期中对铁的需要量较大,易造成缺铁性贫血。如果合理利用铁制剂进行治疗,均能收到满意的疗效。本文用强力铁,肝铁片、硫酸亚铁三种铁制剂进行疗效比较。     

Controlling the Fenton Reaction for Soil Remediation

ABSTRACT

This paper describes the use elemental iron to control the Fenton reaction, a process in which ferrous ion reacts with hydrogen peroxide. It is widely believed that the Fenton reaction produces free radicals that can degrade organic chemicals. By using elemental iron in place of ferrous iron, we found that the vigor of the Fenton reaction can be controlled, and therefore can be used more effectively to remediate contaminated soil.

Laboratory studies were done to compare the elemental iron approach with the original ferrous salt approach. It was found that elemental iron can increase the effectiveness of the Fenton reaction in degrading organic chemical such as Pentachlorophenol (PCP). The mechanism of control lies in the production of ferrous irons from elemental iron in the presence of hydrogen peroxide.     

Formation of protein-coated iron minerals

The ability of iron to cycle between Fe(2+) and Fe(3+) forms has led to the evolution, in different forms, of several iron-containing protein cofactors that are essential for a wide variety of cellular processes, to the extent that virtually all cells require iron for survival and prosperity. The redox properties of iron, however, also mean that this metal is potentially highly toxic and this, coupled with the extreme insolubility of Fe(3+), presents the cell with the significant problem of how to maintain this essential metal in a safe and bioavailable form. This has been overcome through the evolution of proteins capable of reversibly storing iron in the form of a Fe(3+) mineral. For several decades the ferritins have been synonymous with the function of iron storage. Within this family are subfamilies of mammalian, plant and bacterial ferritins which are all composed of 24 subunits assembled to form an essentially spherical protein with a central cavity in which the mineral is laid down. In the past few years it has become clear that other proteins, belonging to the family of DNA-binding proteins from starved cells (the Dps family), which are oligomers of 12 subunits, and to the frataxin family, which may contain up to 48 subunits, are also able to lay down a Fe(3+) mineral core. Here we present an overview of the formation of protein-coated iron minerals, with particular emphasis on the structures of the protein coats and the mechanisms by which they promote core formation. We show on the one hand that significant mechanistic similarities exist between structurally dissimilar proteins, while on the other that relatively small structural differences between otherwise similar proteins result in quite dramatic mechanistic differences.     

Binding of Reactive Oxygen Species at FeS Cubane Clusters

Reactive oxygen species (ROS) play an important role in the biochemistry of the cell and occur in degenerative processes as well as in signal transduction. Iron?sulfur proteins are particularly oxygen‐sensitive and their inorganic cofactors frequently undergo ROS‐induced decomposition reactions. As experimental knowledge about these processes is still incomplete we present here a quantum chemical study of the relative energetics for the binding of the most relevant ROS to [Fe₄S₄] clusters. We find that cubane clusters with one uncoordinated Fe atom (as found, for instance, in aconitase) bind all oxygen derivatives considered, whereas activation of triplet O₂ to singlet O₂ is required for binding to valence‐saturated iron centers in these clusters. The radicals NO and OH feature the most exothermic binding energies to Fe atoms. Direct sulfoxidation of coordinating cysteine residues is only possible by OH or H₂O₂ as attacking agents. The thermodynamic picture of ROS binding to iron?sulfur clusters established here can serve as a starting point for studying reactivity‐modulating effects of the cluster‐embedding protein environment on ROS‐induced decomposition of iron?sulfur proteins.     

Preferential 5‐Methylcytosine Oxidation in the Linker Region of Reconstituted Positioned Nucleosomes by Tet1 Protein

Tet (ten–eleven translocation) family proteins oxidize 5‐methylcytosine (mC) to 5‐hydroxymethylcytosine (hmC), 5‐formylcytosine (fC), and 5‐carboxycytosine (caC), and are suggested to be involved in the active DNA demethylation pathway. In this study, we reconstituted positioned mononucleosomes using CpG‐methylated 382 bp DNA containing the Widom 601 sequence and recombinant histone octamer, and subjected the nucleosome to treatment with Tet1 protein. The sites of oxidized methylcytosine were identified by bisulfite sequencing. We found that, for the oxidation reaction, Tet1 protein prefers mCs located in the linker region of the nucleosome compared with those located in the core region.     

Site‐Specific Oxidation State Assignments of the Iron Atoms in the [4Fe:4S]2+/1+/0 States of the Nitrogenase Fe‐Protein

The nitrogenase iron protein (Fe‐protein) contains an unusual [4Fe:4S] iron‐sulphur cluster that is stable in three oxidation states: 2+, 1+, and 0. Here, we use spatially resolved anomalous dispersion (SpReAD) refinement to determine oxidation assignments for the individual irons for each state. Additionally, we report the 1.13‐Å resolution structure for the ADP bound Fe‐protein, the highest resolution Fe‐protein structure presently determined. In the dithionite‐reduced [4Fe:4S]¹⁺ state, our analysis identifies a solvent exposed, delocalized Fe^2.5+ pair and a buried Fe²⁺ pair. We propose that ATP binding by the Fe‐protein promotes an internal redox rearrangement such that the solvent‐exposed Fe pair becomes reduced, thereby facilitating electron transfer to the nitrogenase molybdenum iron‐protein. In the [4Fe:4S]⁰ and [4Fe:4S]²⁺ states, the SpReAD analysis supports oxidation states assignments for all irons in these clusters of Fe²⁺ and valence delocalized Fe^2.5+, respectively.     

A microfluidic device that generates hydroxyl radicals to probe the solvent accessible surface of nucleic acids

We describe a microfluidic device containing a mineral matrix capable of rapidly generating hydroxyl radicals that enables high-resolution structural studies of nucleic acids. Hydroxyl radicals cleave the solvent accessible backbone of DNA and RNA; the cleavage products can be detected with as fine as single nucleotide resolution. Protection from hydroxyl radical cleavage (footprinting) can identify sites of protein binding or the presence of tertiary structure. Here we report preparation of micron sized particles of iron sulfide (pyrite) and fabrication of a microfluidic prototype that together generate enough hydroxyl radicals within 20 ms to cleave DNA sufficiently for a footprinting analysis to be conducted. This prototype enables the development of high-throughput and/or rapid reaction devices with which to probe nucleic acid folding dynamics and ligand binding.     

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI‐MS

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI‐MS) study of the noncovalent interactions between different intermolecular and intramolecular G‐quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI‐MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results. Copyright © 2008 John Wiley & Sons, Ltd.     

Significance of the molecular shape of iron corrphycene in a protein pocket

The iron complex of a new type of corrphycene bearing two ethoxycarbonyl (-CO2C2H5) groups on the bipyrrole moiety was introduced into apomyoglobin. The reconstituted ferric myoglobin has a coordinating water molecule that deprotonates to hydroxide with a pK(a) value of 7.3 and exhibits 3-10-fold higher affinities for anionic ligands when compared with a counterpart myoglobin with the same substituents on the dipyrroethene moiety. In the ferrous state, the oxygen affinity of the new myoglobin was decreased to 1/410 of the native protein. The anomalies in the ligand binding, notably dependent on the side-chain location, were interpreted in terms of a characteristic core shape of corrphycene that produces the longer and shorter Fe-N(pyrrole) bonds. The spin-state equilibrium analysis of the ferric azide myoglobin containing the new iron corrphycene supported the nonequivalence of the Fe-N(pyrrole) bonds. These results demonstrate that the trapezoidal molecular shape of corrphycene exerts functional significance when the iron complex is placed in a protein pocket.     

Different types of electrospun nanofibers and their effect on microfluidic‐based immunoassay

Protein capturing on polymeric substrate of microfluidic devices is a key factor for the fabrication of immunoassay with high sensitivity. In this work, simple and versatile technique of electrospinning was used to produce electrospun nanofibrous membranes (e.NFMs) with high surface area as a substrate for microfluidic‐based immunoassay to increase sensitivity. It was found that the simultaneous use of e.NFM and 1‐Ethylethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐Hydroxysuccinimide hydroxysuccinimide as coupling agent has synergic effect on antigen immobilization onto the microchannels. It was found that the oxygen plasma technique for the creation of oxygen containing functional group like carboxyl and hydroxyl causes extreme leakage of solution through the microchannels. Thus, due to capillary effect, it is impossible to use hydrophilic substrate to modify microchannels. In order to compensate this problem, it is propose to utilize other type of polymer for the fabrication of nanofiber to answer this important question that if it is possible to enhance the sensitivity of immunoassay just by changing the polymer type? For this purpose, four different polymers, namely, polycaprolactone, poly lactic‐co‐glycolic acid, poly L‐lactic acid, and polyethersolfone were used as the based material for e.NFM fabrication. Results showed that compared with plain poly (dimethylsiloxane) surface of microchannels, poly lactic‐co‐glycolic acidand poly L‐lactic acid, which inherently contain end‐group of carboxyl in their chemical structure, can improve the protein immobilization, which leads to immunoassay signal enhancement through 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide/N‐hydroxysuccinimide coupling chemistry, significantly.     

Investigation on the Photodissociation of Oxygen from Oxymyoglobin by Fluorescence Spectroscopy

Photodissociation of oxygen from oxymyoglobin(oxyMb) was investigated by means of fluorescence spectroscopy. One of the most important findings of the photodissociation of oxyMb was the discovery of two processes which were affected by excitation intensity, temperature, solvent viscosity, and excitation wavelength. Process I(PI) corresponded to oxygen escaping from the binding site at ferrous heme iron atom within the porphyrin ring into the heme pocket, whereas process II(PII) was ascribed to oxygen escaping from the heme pocket into the solvent. To elucidate this interesting phenomenon, we proposed a model that oxygen encountered two barriers on its way from the binding site at the ferrous heme iron to the solvent. Reversibility and wavelength sensitivity of the photodissociation were also observed.     

Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.